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Electrophysiological characterization of a small molecule activator on human ether-a-go-go-related gene (hERG) potassium channel
Journal of Pharmacological Sciences 140 (2019) 284e290
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Electrophysiological characterization of a small molecule activator on human ether-a-go-go-related gene (hERG) potassium channel Xiuming Dong a, 1, Yani Liu a, *, 1, Heling Niu a, Gongxin Wang b, Liying Dong a, b, Anruo Zou a, b, KeWei Wang a, ** a b
Department of Pharmacology, Qingdao University School of Pharmacy, 38 Dengzhou Road, Qingdao, 266021, China Qing Dao Haiwei Biopharma. CO.LTD, 368 Hedong Road, Qingdao, 266000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 February 2019 Received in revised form 30 July 2019 Accepted 1 August 2019 Available online 14 August 2019
The human ether-a-go-go-related gene (hERG) encodes the Kþ channel that carries the rapid component of the delayed rectifier current in the human heart. Reduction of hERG activity induced by gene mutations or pharmacological inhibition is responsible for the type 2 form of long QT syndrome in patients which can develop into ventricular arrhythmia and sudden cardiac death. Therefore, pharmacological activation of hERG may lead to therapeutic potential for cardiac arrhythmias. In this study we characterized a small and novel compound, N-(2-(tert-butyl)phenyl)-6-(4-chlorophenyl)-4-(trifluoromethyl) nicotinamide, HW-0168, that exhibits potent activation of hERG channel with an EC50 of 0.41 ± 0.2 mM. Using whole-cell patch clamp recording of HEK293 cells stably expressed hERG channels, we found that HW-0168 dramatically increased current amplitude about 2.5 folds and slowed down current inactivation about 4 folds. HW-0168 shifted the voltage-dependent channel activation to hyperpolarizing direction about 3.7 mV and the voltage-dependent channel inactivation to depolarizing direction about 9.4 mV. In addition, recording of guinea-pig ventricular cells confirmed that HW-0168 shortened the action potential duration. In conclusion, we identified a novel hERG channel activator HW-0168 that can be used for studying the physiological role of hERG in cardiac myocytes and may be beneficial for treating long QT syndrome. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Activator hERG HW-0168 Whole-cell patch-clamp Long-QT syndrome
1. Introduction Human ether-a-go-go related gene (hERG), also known as KCNH2, encodes for the 11th member of the voltage-gated potassium ion channel subfamily (Kv11.1) that carries the rapid component of the human cardiac delayed rectifier current and plays a significant role in action potential repolarization.1,2 Inhibition of hERG channel function by loss-of-function mutations of hERG gene is associated with the type 2 form of congenital long QT syndrome (LQTS).3,4 In addition, hERG channel is sensitively inhibited by a variety of pharmacological agents, resulting in an acquired form of LQTS.5,6 Both congenital and acquired forms of
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Liu), [email protected] (K. Wang). Peer review under responsibility of Japanese Pharmacological Society. 1 These authors contributed this work equally.
LQTS increase the predisposition for life-threatening arrhythmias in a structurally normal heart.7 Until now, many drugs have been terminated during clinical development or withdrawn from the market due to their side effects on induction of LQTS.8 Therefore, it is necessary to examine the inhibitory effect of pre-clinical compounds on hERG channel for evaluation of their cardiac liability. Physiological or pharmacological potentiating hERG channel function would accelerate action potential repolarization and shorten the duration of action potential.9 Several investigations have identified and characterized pharmacological agents that enhance hERG activity, including RPR260243,10 PD-118057,11,12 PD307243,13 NS1643,14,15 NS3623,16,17 A-935142,18 ICA-105574,19,20 and KB130015.21 The hERG channel activators can increase hERG current via different mechanisms, including the slowdown of voltage-dependent inactivation, deactivation, enhancement of activation or a combination of the above.22 These activators can enhance channel function by accelerating the myocardial repolarization, and also exhibit antiarrhythmic effects in vivo,8 indicating that hERG channel activators might be beneficial for potential
https://doi.org/10.1016/j.jphs.2019.08.001 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Dong et al. / Journal of Pharmacological Sciences 140 (2019) 284e290
therapy for LQTS. hERG activators may also possess developmental potential for treatment of arrhythmia by reducing electrical heterogeneity in the myocardium and re-entry symptoms. In this study, we identified and characterized a novel smallmolecule compound HW-0168. HW-0168 increased hERG current through slowing down the voltage dependent channel inactivation. HW-0168 also shortened the action potential duration of guineapig ventricular myocytes. Therefore, our novel hERG channel opener HW-0168 might be a useful tool for further understanding the role of hERG channel in cardiac repolarization and the pathogenesis of the long-QT syndrome. 2. Materials and methods 2.1. Cell culture The hERG stably transfected HEK293 cells were kindly gifted by Prof. Xuan Zhang (Hebei University of Chinese Medicine, China) and were cultured in DMEM (Gibco, USA) supplemented with 10% Fetal Bovine Serum (Gibco, USA), 400 mg/ml G418 (SigmaeAldrich, USA) and 1% penicillin/streptomycin at 37 C in a humidified atmosphere of air with 5% CO2. For patch-clamp recording, cells were removed from the culture flask by 1-min digestion with 2.5 mg/ml trypsin (1:250) and plated at low density onto 12-mm-diameter glass coverslips in 24-well cell culture plates. The cells were used for recording within 48 h after plating. 2.2. Chemicals N-(2-(tert-butyl)phenyl)-6-(4-chlorophenyl)-4-(trifluoromethyl) nicotinamide, HW-0168 was synthesized at Qingdao University and its chemical structure was verified by MS and NMR analysis. The chemical structure of HW-0168 with molecular mass
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at 439.5 is depicted in Fig. 1A. Dofetilide and albumin from bovine serum (BSA 1 mg/ml) were purchased from Sigma (St. Louis, MO, USA). Collagenase (0.4 mg/ml type II with activity 308 U/mg) was purchased from Worthington (Worthington Biochemicals, USA). 2.3. Isolation of Guinea-Pig ventricular cardiomyocytes Ventricular myocytes were enzymatically isolated from adult male guinea-pigs weighing 200e300 g as described previously.23 Briefly, guinea-pigs were heparinized with 1000 U heparin and anesthetized with barbital sodium (60 mg/kg) by intraperitoneal injection. When skeletal muscle tone was lost, the heart was quickly removed and mounted on a Langendorff apparatus followed by retrograde perfusion with a Tyrode solution containing no calcium (in mM: 132 NaCl, 5.4 KCl, 1 MgCl2, 10 glucose and 10 HEPES, pH 7.4, 100% O2, 37 C) for 5 min. This was subsequently replaced with a solution of the same composition but containing collagenase (0.4 mg/ml type II with activity 308 U/mg, Worthington Biochemicals, USA) and albumin from bovine serum (BSA 1 mg/ml; Sigma, USA). Then, the heart tissue was minced, and myocytes were dispersed in KB solution containing (in mM) 80 KOH, 40 KCl, 25 KH2 PO4, 3 MgSO4, 50 glutamic acid, 20 taurine, 1 EGTA, 10 HEPES and 10 glucose (pH 7.2 with KOH) and stored at 4 C for at least 1 h before use. 2.4. Electrophysiology hERG channel currents were recorded using a MultiClamp 700A amplifier and pCLAMP 10.6 software (Molecular Devices, Sunnyvale, CA) and were filtered at 2 kHz. Patch electrodes with resistances of 2e5 MU were pulled with a horizontal micropipette puller (P-97, Sutter Instruments, USA) and fire polished with MF830 polisher (NARISHIGE, Japan). The extracellular bath solution
Fig. 1. Activation of hERG current by HW-0168. (A) Chemical structure of HW0168: N-(2-(tert-butyl)phenyl)-6-(4-chlorophenyl)-4-(trifluoromethyl) nicotinamide. (B) Representative current traces with (right) or without 1 mM HW-0168 (left) recorded with step protocol at a holding potential of 80 mV before 4-s depolarization to potentials ranging from 60 mV to þ40 mV with 10 mV increment and repolarization to 40 mV for induction of the activation tail currents. (C) Currentevoltage relationship in cells after 5 min exposure to vehicle (black) or HW0-168 (1 mM, red), n ¼ 8, *p < 0.05. (D) Representative current traces recorded at þ10 mV (top, insert) from before (black) and 5 min after exposure to HW-0168 (1 mM, red). The dashed line represents the zero current level.
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(mM): 140 NaCl, 2 MgCl2, 2 CaCl2, 5 KCl, 10 glucose, and 10 HEPES (pH 7.4 adjust with NaOH). The pipette solution (mM): 140 KCl, 2 MgCl2, 5 EGTA and 10 HEPES (pH 7.4 adjust with KOH). HW-0168 was dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution of 10 mM stored at 20 C. The work solution was diluted with the extracellular bath solution. The final concentration of DMSO in the solution was no more than 0.1%, which had no effect on hERG current.
with exponential equations containing one or two components. Data were expressed as the mean ± SEM. Statistical analysis of differences between groups was carried out using Student's paired t-test or unpaired t-test, and p value 0.05 was considered to be statistically significant. 3. Results
2.5. Data analysis
3.1. HW-0168 enhances hERG current amplitude in a voltagedependent manner
All recorded data were analyzed with Clampfit 10.6 (Molecular Devices, USA), OriginPro 8.0 (Origin Lab, USA) and Adobe Illustrator 10 (Adobe, USA). The concentration-response curve was fitted by logistic equation of y ¼ A2þ(A1A2)/(1þ(x/x0)p), where x is the drug concentration, and p is the Hill coefficient. The current activation and inactivation curves were generated by plotting the normalized tail current amplitude against step potentials, and were fitted with a Boltzmann equation of y ¼ A/1 þ exp [(VhVm)/k], where A is the amplitude of relationship, Vh is the voltage for halfmaximal activation, Vm is the test potential, and k is the slope factor of the curve. The inactivation and deactivation traces were fitted
The compound HW-0168 was synthesized by Qingdao University and identified as a hERG channel activator. The chemical structure of HW-0168 is depicted in Fig. 1A. We firstly tested the effect of HW-0168 (1 mM) on currentevoltage relationship of hERG channels stably expressed in HEK293 cells. The cells were voltage clamped to a holding potential of 80 mV and followed by 4-s depolarization to potentials ranging from 60 mV to þ40 mV with 10 mV increment before repolarized to 40 mV for induction of activation tail current. HW-0168 (1 mM) significantly increased the hERG current amplitude (Fig. 1B). The IeV relationship for hERG current measured at the end of 4-s pre-test pulse with or without
Fig. 2. Concentration-dependent activation of hERG current by HW-0168. (A) Representative current traces induced by voltage step at þ10 mV (from a holding potential of 80 mV) during the application of different concentrations (as indicated) of HW-0168. The dashed line represents the zero current level. (B) Concentration-effect curve of HW0168 was fitted by logistic function with EC50 of 0.41 ± 0.2 mM, n ¼ 4e10.
Fig. 3. Voltage-dependent activation and inactivation of hERG currents by HW-0168. (A) Normalized of voltage-dependent activation of hERG currents generated from activation tail currents measured at the second 40 mV before (control, square) and after HW-0168 (1 mM, round) and fitted with Boltzmann function. HW-0168 (red) caused a hyperpolarization shift in V1/2 to 21.3 mV from the control (black) value of 17.6 mV. (B) Normalized of voltage-dependent inactivation of hERG currents generated from tail currents measured at the second þ40 mV before (control, square) and after HW-0168 (1 mM, round) and fitted with Boltzmann function. HW-0168 (red) caused a depolarization shift in V1/2 to 71.1 mV from the control (black) value of 80.5 mV. Each data point is expressed as the mean ± SEM.
X. Dong et al. / Journal of Pharmacological Sciences 140 (2019) 284e290
1 mM HW-0168 was summarized in Fig. 1C (n ¼ 8). The hERG current measured at þ10 mV was enhanced by ~2.5-fold when 1 mM HW-0168 was perfused to HEK293 cells expressed hERG channels (Fig. 1D). We next investigated the concentration dependent effects of HW-0168 on hERG currents. As shown in Fig. 2, HW-0168 activated hERG channel in a concentration-dependent manner with an EC50 of 0.41 ± 0.2 mM (n ¼ 4e10). We further evaluated the effect of HW-0168 on voltage dependent activation and inactivation of hERG current. The currents were recorded using a voltage step protocol as indicated in Fig. 3A, insert. The activation tail currents at 40 mV were obtained and fitted
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with Boltzmann function. As shown in Fig. 3A, perfusion of 1 mM HW-0168 showed no obvious shift on hERG activation curve with half-maximal activation voltage (V1/2) at 17.6 mV (control), as compared with 21.3 mV (HW-0168). We also tested the effects of different concentrations of HW-0168 on hERG channel activation, and no obvious dose-dependent voltage-shifts were seen (Data not shown). We next also investigated the effect of HW-0168 on hERG channel inactivation. As shown in Fig. 3B, perfusion of 1 mM HW0168 significantly shifted the inactivation of hERG channel to more depolarizing membrane potential at 71.1 ± 2.7 mV, as compared with 80.5 ± 1.4 mV in control.
Fig. 4. Slowness of hERG inactivation rate by HW-0168. (A) Cells were held at 80 mV and then depolarized to þ40 mV for 1-s to fully activate and inactivate hERG channel current before repolarized to 100 mV for 6 ms for channel recovery from inactivation. A third pulse was applied to a potential that was varied from 20 to þ70 mV with 10 mV increment to observe the re-onset of current inactivation. Representative current traces upon each depolarizing voltage in the absence (control, black) or presence of 1 mM HW-0168 (red) were shown. (B) Representative deactivation current traces recorded at þ70 mV in the absence (control, black) or presence of 1 mM HW-0168 (red). (C) The time constants of inactivation at each corresponding voltage fitted with single exponential equation (n ¼ 5). Each data point is expressed as the mean ± SEM and *p < 0.05, *p < 0.01, and ***p < 0.001 indicates statistical difference.
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3.2. HW-0168 slows down the rate of inactivation but not deactivation The onset rate of hERG current inactivation was determined using a three-pulse protocol as previously described.24 The hERG channel was fully activated and inactivated by a 1-s pulse to þ40 mV before repolarized to 100 mV for 6 ms for channel recovery from inactivation. A third pulse was applied to a potential that was varied from 20 to þ70 mV with 10 mV increment to observe the re-onset of current inactivation (Fig. 4A, top panel). The current traces upon each depolarizing voltage in the absence or presence of 1 mM HW-0168 are detected (Fig. 4A, middle and bottom panels). The currents obtained from variable potentials were fitted to single exponential function for obtaining the time constants of inactivation, which was plotted versus the repolarizing voltages. HW-0168 at 1 mM significantly slowed the rate of hERG inactivation (Fig. 4C, n ¼ 5). The time constant of inactivation is 5.95 ± 0.48 ms in the presence of HW-0168 compared with 1.49 ± 0.16 ms in the absence of HW-0168 fitted at þ70 mV, which is about 4 folds slower. To assess the effect of HW-0168 on deactivation, we recorded the cells depolarized to þ20 mV for 500 ms before repolarizing to
different potentials from 120 mV to 40 mV with 10 mV increment for generation of inward tail currents. Deactivation was determined by fitting these tail currents to a double exponential function. As shown in Fig. 5A, the tail currents were obtained in the presence and absence of 1 mM HW-0168 that produced no significant effects on the rate of deactivation at tested potentials (Fig. 5C and D). 3.3. Shortening of cardiac action potential duration (APD) by HW0168 The enhancing effect of hERG by HW-0168 suggested that HW0168 might shorten the action potential duration of native cardiac myocytes. To verify this notion, we recorded action potentials of guinea-pig ventricular myocytes using the perforated-whole cell patch technique under current-clamping mode. As shown in Fig. 6, the evoked action potential was elicited by injection of 1 nA current into the cell, HW-0168 (1 mM) caused shortening of APD and could be easily washed out, as compared with dofetilide (1 mM) that induced an obvious increased APD. The shortening of APD by HW0168 suggests that HW-0168 might be effective in treating druginduced LQT syndrome.
Fig. 5. Effect of HW-0168 on hERG deactivation. (A) Cells were held at 80 mV and depolarized to þ20 mV for 500 ms before repolarized to different potentials from 120 mV to 40 mV with 10 mV increment for generation of inward tail currents as indicated at the top of panel A. Representative current traces upon each depolarizing voltage in the absence or presence of 1 mM HW-0168 are shown at bottom of panel A. (B) Representative current traces were obtained at 120 mV in the absence (control, black) or presence of 1 mM HW-0168 (red). (C) and (D) Time constants (t) for fast and slow components of current deactivation in the absence (control) and presence of HW-0168 (1 mM) fitted with double exponential equation. Data were expressed as the mean ± SEM (n ¼ 5).
X. Dong et al. / Journal of Pharmacological Sciences 140 (2019) 284e290
Fig. 6. HW-0168 reduces action potential duration in guinea-pig ventricular cardiomyocytes. Isolated Guinea-pig ventricular cardiomyocytes were recorded for action potential using perforated whole cell patch clamp technique under current-clamping mode. Action potentials in cells were induced by injection of a depolarizing current of 1 nA. Representative evoked action potentials in the absence (control, green; wash, red) or presence of different compounds (HW-0168, black; Dofetilide, blue) as indicated. The dashed line represents the zero voltage level.
4. Discussion Drug-induced LQTS has been particularly investigated due to the fact that it delayed repolarization of ventricular action potentials and is related to a potentially fatal cardiac arrhythmia (Torsades de Pointes) which can cause sudden deaths. Until now, the therapeutic strategies for LQTS include pharmacologic treatment and surgery with implanted devices. However, these treatments are not always effective and also cause other side effects.25e27 Activators of hERG channel could reverse hERG blockers' effect and might be a new pharmacological approach for therapy of drug-induced LQTS. However, no effective activators of hERG channel are discovered for LQTS treatment so far. We identified and characterized a novel small molecule compound HW-0168 which exhibits potent activation of hERG potassium channel. HW-0168 at 1 mM enhances hERG current about 2.5 folds at þ10 mV on HEK293 cells stably expressing hERG channel. The enhancement on voltage dependent activation of hERG current resemble the effects reported for majority of hERG openers. Although the hyperpolarizing shifts (up to 3.7 mV) in voltagedependent channel activation and depolarizing shifts (up to 9.4 mV) in voltage-dependent channel inactivation generated by HW-0168 (1 mM) are quite limited compared with compounds such as RPR260243,10 PD-307243,13 NS1643,28 NS3623,16 A-93514229 that produce depolarizing shifts about 15e35 mV, and even ICA105574 that causes depolarizing shift over 180 mV in voltage dependent inactivation.19 However, it is not clear whether the mechanism of HW-0168 is consistent with the reported compounds and it needs further exploration. Our results show that HW-0168 activates hERG channel with an EC50 value of 0.41 ± 0.2 mM and is much potent than some other reported hERG activators, such as NS3623 with an EC50 value of 79.4 mM,16 ICA-105574 with an EC50 value of 0.5 mM,19 KB130015 with an EC50 value of 12 mM.30 However, compared with reported compounds, the effective window of HW-0168 is comparatively limited and the activation effect is reduced as the concentration goes higher than 1 mM. In addition, the mechanism of HW-0168 to enhance hERG channel activity is primarily involved in slowing onset of hERG current inactivation. This enhancement is voltagedependent, being more pronounced at more depolarized potentials, which is similar to NS3623 and NS1643.16 No effect of HW0168 on hERG deactivation was observed in this study, which is
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differentiated itself from some other hERG openers, such as RPR260243, ICA-105574 and A-935142. RPR260243 dramatically slows current deactivation in a temperature- and voltagedependent manner.10 A-935142 slows hERG channel deactivation at multiple voltage potentials.18 Meanwhile, we investigated and confirmed the shortening effect of HW-0168 on action potentials in guinea-pig ventricular myocytes by using current-clamping technique. Some other hERG channel activators such as RPR260243 that can increase the T-wave amplitude, prolong the PR interval and shorten the QT interval in guinea-pig hearts; and PD-118057 that shortens the action potential duration and prevents QT prolongation by dofetilide.10,31 HW-0168 has similar effects compared with these reported compounds, suggesting that HW-0168 might be effective in treating drug-induced LQTS although the mechanism of HW-0168 requires further investigations. In summary, HW-0168 as an opener of hREG channels is more potent than most of activators reported. The structure and function of HW-0168 might inspire further development of more potent and specific agents that can be beneficial for antiarrhythmic therapy. Conflict of interests All authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This work is supported by research grants awarded to WKW from the National Natural Science Foundation of China, China (81573410), the Ministry of Science and Technology of China, China (2018ZX09711001-004-006) and the Natural Sciences Foundation of Shandong Province, China (ZR2015QL008). We are grateful to the gift of hERG stable HEK293 cells from Prof. Xuan Zhang (Hebei University of Chinese Medicine, China). References 1. Fujii S, Hirota A, Kamino K. Optical recording of development of electrical activity in embryonic chick heart during early phases of cardiogenesis. J Physiol. 1981;311:147e160. 2. Warmke JW, Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci U S A. 1994;91(8):3438e3442. 3. Osterbur Badhey ML, Bertalovitz AC, McDonald TV. Express with caution: epitope tags and cDNA variants effects on hERG channel trafficking, half-life and function. J Cardiovasc Electrophysiol. 2017;28(9):1070e1082. 4. Sakaguchi T, Itoh H, Ding WG, et al. Hydroxyzine, a first generation H(1)receptor antagonist, inhibits human ether-a-go-go-related gene (HERG) current and causes syncope in a patient with the HERG mutation. J Pharmacol Sci. 2008;108(4):462e471. 5. Lu HR, Hermans AN, Gallacher DJ. Does terfenadine-induced ventricular tachycardia/fibrillation directly relate to its QT prolongation and Torsades de Pointes? Br J Pharmacol. 2012;166(4):1490e1502. 6. Dennis AT, Wang L, Wan H, Nassal D, Deschenes I, Ficker E. Molecular determinants of pentamidine-induced hERG trafficking inhibition. Mol Pharmacol. 2012;81(2):198e209. 7. Fu DG. Cardiac arrhythmias: diagnosis, symptoms, and treatments. Cell Biochem Biophys. 2015;73(2):291e296. 8. Foo B, Williamson B, Young JC, Lukacs G, Shrier A. hERG quality control and the long QT syndrome. J Physiol. 2016;594(9):2469e2481. 9. Zhou PZ, Babcock J, Liu LQ, Li M, Gao ZB. Activation of human ether-a-go-go related gene (hERG) potassium channels by small molecules. Acta Pharmacol Sin. 2011;32(6):781e788. 10. Kang J, Chen XL, Wang H, et al. Discovery of a small molecule activator of the human ether-a-go-go-related gene (HERG) cardiac Kþ channel. Mol Pharmacol. 2005;67(3):827e836. 11. Zhou J, Augelli-Szafran CE, Bradley JA, et al. Novel potent human ether-a-gogo-related gene (hERG) potassium channel enhancers and their in vitro antiarrhythmic activity. Mol Pharmacol. 2005;68(3):876e884. 12. Perry M, Sachse FB, Abbruzzese J, Sanguinetti MC. PD-118057 contacts the pore helix of hERG1 channels to attenuate inactivation and enhance Kþ conductance. Proc Natl Acad Sci U S A. 2009;106(47):20075e20080. 13. Gordon E, Lozinskaya IM, Lin Z, et al. 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro1H-isoindol-5-ylamino]-nicotinic acid (PD-307243) causes instantaneous
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Full Paper
Electrophysiological characterization of a small molecule activator on human ether-a-go-go-related gene (hERG) potassium channel Xiuming Dong a, 1, Yani Liu a, *, 1, Heling Niu a, Gongxin Wang b, Liying Dong a, b, Anruo Zou a, b, KeWei Wang a, ** a b
Department of Pharmacology, Qingdao University School of Pharmacy, 38 Dengzhou Road, Qingdao, 266021, China Qing Dao Haiwei Biopharma. CO.LTD, 368 Hedong Road, Qingdao, 266000, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 February 2019 Received in revised form 30 July 2019 Accepted 1 August 2019 Available online 14 August 2019
The human ether-a-go-go-related gene (hERG) encodes the Kþ channel that carries the rapid component of the delayed rectifier current in the human heart. Reduction of hERG activity induced by gene mutations or pharmacological inhibition is responsible for the type 2 form of long QT syndrome in patients which can develop into ventricular arrhythmia and sudden cardiac death. Therefore, pharmacological activation of hERG may lead to therapeutic potential for cardiac arrhythmias. In this study we characterized a small and novel compound, N-(2-(tert-butyl)phenyl)-6-(4-chlorophenyl)-4-(trifluoromethyl) nicotinamide, HW-0168, that exhibits potent activation of hERG channel with an EC50 of 0.41 ± 0.2 mM. Using whole-cell patch clamp recording of HEK293 cells stably expressed hERG channels, we found that HW-0168 dramatically increased current amplitude about 2.5 folds and slowed down current inactivation about 4 folds. HW-0168 shifted the voltage-dependent channel activation to hyperpolarizing direction about 3.7 mV and the voltage-dependent channel inactivation to depolarizing direction about 9.4 mV. In addition, recording of guinea-pig ventricular cells confirmed that HW-0168 shortened the action potential duration. In conclusion, we identified a novel hERG channel activator HW-0168 that can be used for studying the physiological role of hERG in cardiac myocytes and may be beneficial for treating long QT syndrome. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Activator hERG HW-0168 Whole-cell patch-clamp Long-QT syndrome
1. Introduction Human ether-a-go-go related gene (hERG), also known as KCNH2, encodes for the 11th member of the voltage-gated potassium ion channel subfamily (Kv11.1) that carries the rapid component of the human cardiac delayed rectifier current and plays a significant role in action potential repolarization.1,2 Inhibition of hERG channel function by loss-of-function mutations of hERG gene is associated with the type 2 form of congenital long QT syndrome (LQTS).3,4 In addition, hERG channel is sensitively inhibited by a variety of pharmacological agents, resulting in an acquired form of LQTS.5,6 Both congenital and acquired forms of
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Liu), [email protected] (K. Wang). Peer review under responsibility of Japanese Pharmacological Society. 1 These authors contributed this work equally.
LQTS increase the predisposition for life-threatening arrhythmias in a structurally normal heart.7 Until now, many drugs have been terminated during clinical development or withdrawn from the market due to their side effects on induction of LQTS.8 Therefore, it is necessary to examine the inhibitory effect of pre-clinical compounds on hERG channel for evaluation of their cardiac liability. Physiological or pharmacological potentiating hERG channel function would accelerate action potential repolarization and shorten the duration of action potential.9 Several investigations have identified and characterized pharmacological agents that enhance hERG activity, including RPR260243,10 PD-118057,11,12 PD307243,13 NS1643,14,15 NS3623,16,17 A-935142,18 ICA-105574,19,20 and KB130015.21 The hERG channel activators can increase hERG current via different mechanisms, including the slowdown of voltage-dependent inactivation, deactivation, enhancement of activation or a combination of the above.22 These activators can enhance channel function by accelerating the myocardial repolarization, and also exhibit antiarrhythmic effects in vivo,8 indicating that hERG channel activators might be beneficial for potential
https://doi.org/10.1016/j.jphs.2019.08.001 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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therapy for LQTS. hERG activators may also possess developmental potential for treatment of arrhythmia by reducing electrical heterogeneity in the myocardium and re-entry symptoms. In this study, we identified and characterized a novel smallmolecule compound HW-0168. HW-0168 increased hERG current through slowing down the voltage dependent channel inactivation. HW-0168 also shortened the action potential duration of guineapig ventricular myocytes. Therefore, our novel hERG channel opener HW-0168 might be a useful tool for further understanding the role of hERG channel in cardiac repolarization and the pathogenesis of the long-QT syndrome. 2. Materials and methods 2.1. Cell culture The hERG stably transfected HEK293 cells were kindly gifted by Prof. Xuan Zhang (Hebei University of Chinese Medicine, China) and were cultured in DMEM (Gibco, USA) supplemented with 10% Fetal Bovine Serum (Gibco, USA), 400 mg/ml G418 (SigmaeAldrich, USA) and 1% penicillin/streptomycin at 37 C in a humidified atmosphere of air with 5% CO2. For patch-clamp recording, cells were removed from the culture flask by 1-min digestion with 2.5 mg/ml trypsin (1:250) and plated at low density onto 12-mm-diameter glass coverslips in 24-well cell culture plates. The cells were used for recording within 48 h after plating. 2.2. Chemicals N-(2-(tert-butyl)phenyl)-6-(4-chlorophenyl)-4-(trifluoromethyl) nicotinamide, HW-0168 was synthesized at Qingdao University and its chemical structure was verified by MS and NMR analysis. The chemical structure of HW-0168 with molecular mass
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at 439.5 is depicted in Fig. 1A. Dofetilide and albumin from bovine serum (BSA 1 mg/ml) were purchased from Sigma (St. Louis, MO, USA). Collagenase (0.4 mg/ml type II with activity 308 U/mg) was purchased from Worthington (Worthington Biochemicals, USA). 2.3. Isolation of Guinea-Pig ventricular cardiomyocytes Ventricular myocytes were enzymatically isolated from adult male guinea-pigs weighing 200e300 g as described previously.23 Briefly, guinea-pigs were heparinized with 1000 U heparin and anesthetized with barbital sodium (60 mg/kg) by intraperitoneal injection. When skeletal muscle tone was lost, the heart was quickly removed and mounted on a Langendorff apparatus followed by retrograde perfusion with a Tyrode solution containing no calcium (in mM: 132 NaCl, 5.4 KCl, 1 MgCl2, 10 glucose and 10 HEPES, pH 7.4, 100% O2, 37 C) for 5 min. This was subsequently replaced with a solution of the same composition but containing collagenase (0.4 mg/ml type II with activity 308 U/mg, Worthington Biochemicals, USA) and albumin from bovine serum (BSA 1 mg/ml; Sigma, USA). Then, the heart tissue was minced, and myocytes were dispersed in KB solution containing (in mM) 80 KOH, 40 KCl, 25 KH2 PO4, 3 MgSO4, 50 glutamic acid, 20 taurine, 1 EGTA, 10 HEPES and 10 glucose (pH 7.2 with KOH) and stored at 4 C for at least 1 h before use. 2.4. Electrophysiology hERG channel currents were recorded using a MultiClamp 700A amplifier and pCLAMP 10.6 software (Molecular Devices, Sunnyvale, CA) and were filtered at 2 kHz. Patch electrodes with resistances of 2e5 MU were pulled with a horizontal micropipette puller (P-97, Sutter Instruments, USA) and fire polished with MF830 polisher (NARISHIGE, Japan). The extracellular bath solution
Fig. 1. Activation of hERG current by HW-0168. (A) Chemical structure of HW0168: N-(2-(tert-butyl)phenyl)-6-(4-chlorophenyl)-4-(trifluoromethyl) nicotinamide. (B) Representative current traces with (right) or without 1 mM HW-0168 (left) recorded with step protocol at a holding potential of 80 mV before 4-s depolarization to potentials ranging from 60 mV to þ40 mV with 10 mV increment and repolarization to 40 mV for induction of the activation tail currents. (C) Currentevoltage relationship in cells after 5 min exposure to vehicle (black) or HW0-168 (1 mM, red), n ¼ 8, *p < 0.05. (D) Representative current traces recorded at þ10 mV (top, insert) from before (black) and 5 min after exposure to HW-0168 (1 mM, red). The dashed line represents the zero current level.
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(mM): 140 NaCl, 2 MgCl2, 2 CaCl2, 5 KCl, 10 glucose, and 10 HEPES (pH 7.4 adjust with NaOH). The pipette solution (mM): 140 KCl, 2 MgCl2, 5 EGTA and 10 HEPES (pH 7.4 adjust with KOH). HW-0168 was dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution of 10 mM stored at 20 C. The work solution was diluted with the extracellular bath solution. The final concentration of DMSO in the solution was no more than 0.1%, which had no effect on hERG current.
with exponential equations containing one or two components. Data were expressed as the mean ± SEM. Statistical analysis of differences between groups was carried out using Student's paired t-test or unpaired t-test, and p value 0.05 was considered to be statistically significant. 3. Results
2.5. Data analysis
3.1. HW-0168 enhances hERG current amplitude in a voltagedependent manner
All recorded data were analyzed with Clampfit 10.6 (Molecular Devices, USA), OriginPro 8.0 (Origin Lab, USA) and Adobe Illustrator 10 (Adobe, USA). The concentration-response curve was fitted by logistic equation of y ¼ A2þ(A1A2)/(1þ(x/x0)p), where x is the drug concentration, and p is the Hill coefficient. The current activation and inactivation curves were generated by plotting the normalized tail current amplitude against step potentials, and were fitted with a Boltzmann equation of y ¼ A/1 þ exp [(VhVm)/k], where A is the amplitude of relationship, Vh is the voltage for halfmaximal activation, Vm is the test potential, and k is the slope factor of the curve. The inactivation and deactivation traces were fitted
The compound HW-0168 was synthesized by Qingdao University and identified as a hERG channel activator. The chemical structure of HW-0168 is depicted in Fig. 1A. We firstly tested the effect of HW-0168 (1 mM) on currentevoltage relationship of hERG channels stably expressed in HEK293 cells. The cells were voltage clamped to a holding potential of 80 mV and followed by 4-s depolarization to potentials ranging from 60 mV to þ40 mV with 10 mV increment before repolarized to 40 mV for induction of activation tail current. HW-0168 (1 mM) significantly increased the hERG current amplitude (Fig. 1B). The IeV relationship for hERG current measured at the end of 4-s pre-test pulse with or without
Fig. 2. Concentration-dependent activation of hERG current by HW-0168. (A) Representative current traces induced by voltage step at þ10 mV (from a holding potential of 80 mV) during the application of different concentrations (as indicated) of HW-0168. The dashed line represents the zero current level. (B) Concentration-effect curve of HW0168 was fitted by logistic function with EC50 of 0.41 ± 0.2 mM, n ¼ 4e10.
Fig. 3. Voltage-dependent activation and inactivation of hERG currents by HW-0168. (A) Normalized of voltage-dependent activation of hERG currents generated from activation tail currents measured at the second 40 mV before (control, square) and after HW-0168 (1 mM, round) and fitted with Boltzmann function. HW-0168 (red) caused a hyperpolarization shift in V1/2 to 21.3 mV from the control (black) value of 17.6 mV. (B) Normalized of voltage-dependent inactivation of hERG currents generated from tail currents measured at the second þ40 mV before (control, square) and after HW-0168 (1 mM, round) and fitted with Boltzmann function. HW-0168 (red) caused a depolarization shift in V1/2 to 71.1 mV from the control (black) value of 80.5 mV. Each data point is expressed as the mean ± SEM.
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1 mM HW-0168 was summarized in Fig. 1C (n ¼ 8). The hERG current measured at þ10 mV was enhanced by ~2.5-fold when 1 mM HW-0168 was perfused to HEK293 cells expressed hERG channels (Fig. 1D). We next investigated the concentration dependent effects of HW-0168 on hERG currents. As shown in Fig. 2, HW-0168 activated hERG channel in a concentration-dependent manner with an EC50 of 0.41 ± 0.2 mM (n ¼ 4e10). We further evaluated the effect of HW-0168 on voltage dependent activation and inactivation of hERG current. The currents were recorded using a voltage step protocol as indicated in Fig. 3A, insert. The activation tail currents at 40 mV were obtained and fitted
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with Boltzmann function. As shown in Fig. 3A, perfusion of 1 mM HW-0168 showed no obvious shift on hERG activation curve with half-maximal activation voltage (V1/2) at 17.6 mV (control), as compared with 21.3 mV (HW-0168). We also tested the effects of different concentrations of HW-0168 on hERG channel activation, and no obvious dose-dependent voltage-shifts were seen (Data not shown). We next also investigated the effect of HW-0168 on hERG channel inactivation. As shown in Fig. 3B, perfusion of 1 mM HW0168 significantly shifted the inactivation of hERG channel to more depolarizing membrane potential at 71.1 ± 2.7 mV, as compared with 80.5 ± 1.4 mV in control.
Fig. 4. Slowness of hERG inactivation rate by HW-0168. (A) Cells were held at 80 mV and then depolarized to þ40 mV for 1-s to fully activate and inactivate hERG channel current before repolarized to 100 mV for 6 ms for channel recovery from inactivation. A third pulse was applied to a potential that was varied from 20 to þ70 mV with 10 mV increment to observe the re-onset of current inactivation. Representative current traces upon each depolarizing voltage in the absence (control, black) or presence of 1 mM HW-0168 (red) were shown. (B) Representative deactivation current traces recorded at þ70 mV in the absence (control, black) or presence of 1 mM HW-0168 (red). (C) The time constants of inactivation at each corresponding voltage fitted with single exponential equation (n ¼ 5). Each data point is expressed as the mean ± SEM and *p < 0.05, *p < 0.01, and ***p < 0.001 indicates statistical difference.
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3.2. HW-0168 slows down the rate of inactivation but not deactivation The onset rate of hERG current inactivation was determined using a three-pulse protocol as previously described.24 The hERG channel was fully activated and inactivated by a 1-s pulse to þ40 mV before repolarized to 100 mV for 6 ms for channel recovery from inactivation. A third pulse was applied to a potential that was varied from 20 to þ70 mV with 10 mV increment to observe the re-onset of current inactivation (Fig. 4A, top panel). The current traces upon each depolarizing voltage in the absence or presence of 1 mM HW-0168 are detected (Fig. 4A, middle and bottom panels). The currents obtained from variable potentials were fitted to single exponential function for obtaining the time constants of inactivation, which was plotted versus the repolarizing voltages. HW-0168 at 1 mM significantly slowed the rate of hERG inactivation (Fig. 4C, n ¼ 5). The time constant of inactivation is 5.95 ± 0.48 ms in the presence of HW-0168 compared with 1.49 ± 0.16 ms in the absence of HW-0168 fitted at þ70 mV, which is about 4 folds slower. To assess the effect of HW-0168 on deactivation, we recorded the cells depolarized to þ20 mV for 500 ms before repolarizing to
different potentials from 120 mV to 40 mV with 10 mV increment for generation of inward tail currents. Deactivation was determined by fitting these tail currents to a double exponential function. As shown in Fig. 5A, the tail currents were obtained in the presence and absence of 1 mM HW-0168 that produced no significant effects on the rate of deactivation at tested potentials (Fig. 5C and D). 3.3. Shortening of cardiac action potential duration (APD) by HW0168 The enhancing effect of hERG by HW-0168 suggested that HW0168 might shorten the action potential duration of native cardiac myocytes. To verify this notion, we recorded action potentials of guinea-pig ventricular myocytes using the perforated-whole cell patch technique under current-clamping mode. As shown in Fig. 6, the evoked action potential was elicited by injection of 1 nA current into the cell, HW-0168 (1 mM) caused shortening of APD and could be easily washed out, as compared with dofetilide (1 mM) that induced an obvious increased APD. The shortening of APD by HW0168 suggests that HW-0168 might be effective in treating druginduced LQT syndrome.
Fig. 5. Effect of HW-0168 on hERG deactivation. (A) Cells were held at 80 mV and depolarized to þ20 mV for 500 ms before repolarized to different potentials from 120 mV to 40 mV with 10 mV increment for generation of inward tail currents as indicated at the top of panel A. Representative current traces upon each depolarizing voltage in the absence or presence of 1 mM HW-0168 are shown at bottom of panel A. (B) Representative current traces were obtained at 120 mV in the absence (control, black) or presence of 1 mM HW-0168 (red). (C) and (D) Time constants (t) for fast and slow components of current deactivation in the absence (control) and presence of HW-0168 (1 mM) fitted with double exponential equation. Data were expressed as the mean ± SEM (n ¼ 5).
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Fig. 6. HW-0168 reduces action potential duration in guinea-pig ventricular cardiomyocytes. Isolated Guinea-pig ventricular cardiomyocytes were recorded for action potential using perforated whole cell patch clamp technique under current-clamping mode. Action potentials in cells were induced by injection of a depolarizing current of 1 nA. Representative evoked action potentials in the absence (control, green; wash, red) or presence of different compounds (HW-0168, black; Dofetilide, blue) as indicated. The dashed line represents the zero voltage level.
4. Discussion Drug-induced LQTS has been particularly investigated due to the fact that it delayed repolarization of ventricular action potentials and is related to a potentially fatal cardiac arrhythmia (Torsades de Pointes) which can cause sudden deaths. Until now, the therapeutic strategies for LQTS include pharmacologic treatment and surgery with implanted devices. However, these treatments are not always effective and also cause other side effects.25e27 Activators of hERG channel could reverse hERG blockers' effect and might be a new pharmacological approach for therapy of drug-induced LQTS. However, no effective activators of hERG channel are discovered for LQTS treatment so far. We identified and characterized a novel small molecule compound HW-0168 which exhibits potent activation of hERG potassium channel. HW-0168 at 1 mM enhances hERG current about 2.5 folds at þ10 mV on HEK293 cells stably expressing hERG channel. The enhancement on voltage dependent activation of hERG current resemble the effects reported for majority of hERG openers. Although the hyperpolarizing shifts (up to 3.7 mV) in voltagedependent channel activation and depolarizing shifts (up to 9.4 mV) in voltage-dependent channel inactivation generated by HW-0168 (1 mM) are quite limited compared with compounds such as RPR260243,10 PD-307243,13 NS1643,28 NS3623,16 A-93514229 that produce depolarizing shifts about 15e35 mV, and even ICA105574 that causes depolarizing shift over 180 mV in voltage dependent inactivation.19 However, it is not clear whether the mechanism of HW-0168 is consistent with the reported compounds and it needs further exploration. Our results show that HW-0168 activates hERG channel with an EC50 value of 0.41 ± 0.2 mM and is much potent than some other reported hERG activators, such as NS3623 with an EC50 value of 79.4 mM,16 ICA-105574 with an EC50 value of 0.5 mM,19 KB130015 with an EC50 value of 12 mM.30 However, compared with reported compounds, the effective window of HW-0168 is comparatively limited and the activation effect is reduced as the concentration goes higher than 1 mM. In addition, the mechanism of HW-0168 to enhance hERG channel activity is primarily involved in slowing onset of hERG current inactivation. This enhancement is voltagedependent, being more pronounced at more depolarized potentials, which is similar to NS3623 and NS1643.16 No effect of HW0168 on hERG deactivation was observed in this study, which is
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differentiated itself from some other hERG openers, such as RPR260243, ICA-105574 and A-935142. RPR260243 dramatically slows current deactivation in a temperature- and voltagedependent manner.10 A-935142 slows hERG channel deactivation at multiple voltage potentials.18 Meanwhile, we investigated and confirmed the shortening effect of HW-0168 on action potentials in guinea-pig ventricular myocytes by using current-clamping technique. Some other hERG channel activators such as RPR260243 that can increase the T-wave amplitude, prolong the PR interval and shorten the QT interval in guinea-pig hearts; and PD-118057 that shortens the action potential duration and prevents QT prolongation by dofetilide.10,31 HW-0168 has similar effects compared with these reported compounds, suggesting that HW-0168 might be effective in treating drug-induced LQTS although the mechanism of HW-0168 requires further investigations. In summary, HW-0168 as an opener of hREG channels is more potent than most of activators reported. The structure and function of HW-0168 might inspire further development of more potent and specific agents that can be beneficial for antiarrhythmic therapy. Conflict of interests All authors declare that there is no conflict of interests regarding the publication of this paper. Acknowledgments This work is supported by research grants awarded to WKW from the National Natural Science Foundation of China, China (81573410), the Ministry of Science and Technology of China, China (2018ZX09711001-004-006) and the Natural Sciences Foundation of Shandong Province, China (ZR2015QL008). We are grateful to the gift of hERG stable HEK293 cells from Prof. Xuan Zhang (Hebei University of Chinese Medicine, China). References 1. Fujii S, Hirota A, Kamino K. Optical recording of development of electrical activity in embryonic chick heart during early phases of cardiogenesis. J Physiol. 1981;311:147e160. 2. Warmke JW, Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci U S A. 1994;91(8):3438e3442. 3. Osterbur Badhey ML, Bertalovitz AC, McDonald TV. Express with caution: epitope tags and cDNA variants effects on hERG channel trafficking, half-life and function. J Cardiovasc Electrophysiol. 2017;28(9):1070e1082. 4. Sakaguchi T, Itoh H, Ding WG, et al. Hydroxyzine, a first generation H(1)receptor antagonist, inhibits human ether-a-go-go-related gene (HERG) current and causes syncope in a patient with the HERG mutation. J Pharmacol Sci. 2008;108(4):462e471. 5. Lu HR, Hermans AN, Gallacher DJ. Does terfenadine-induced ventricular tachycardia/fibrillation directly relate to its QT prolongation and Torsades de Pointes? Br J Pharmacol. 2012;166(4):1490e1502. 6. Dennis AT, Wang L, Wan H, Nassal D, Deschenes I, Ficker E. Molecular determinants of pentamidine-induced hERG trafficking inhibition. Mol Pharmacol. 2012;81(2):198e209. 7. Fu DG. Cardiac arrhythmias: diagnosis, symptoms, and treatments. Cell Biochem Biophys. 2015;73(2):291e296. 8. Foo B, Williamson B, Young JC, Lukacs G, Shrier A. hERG quality control and the long QT syndrome. J Physiol. 2016;594(9):2469e2481. 9. Zhou PZ, Babcock J, Liu LQ, Li M, Gao ZB. Activation of human ether-a-go-go related gene (hERG) potassium channels by small molecules. Acta Pharmacol Sin. 2011;32(6):781e788. 10. Kang J, Chen XL, Wang H, et al. Discovery of a small molecule activator of the human ether-a-go-go-related gene (HERG) cardiac Kþ channel. Mol Pharmacol. 2005;67(3):827e836. 11. Zhou J, Augelli-Szafran CE, Bradley JA, et al. Novel potent human ether-a-gogo-related gene (hERG) potassium channel enhancers and their in vitro antiarrhythmic activity. Mol Pharmacol. 2005;68(3):876e884. 12. Perry M, Sachse FB, Abbruzzese J, Sanguinetti MC. PD-118057 contacts the pore helix of hERG1 channels to attenuate inactivation and enhance Kþ conductance. Proc Natl Acad Sci U S A. 2009;106(47):20075e20080. 13. Gordon E, Lozinskaya IM, Lin Z, et al. 2-[2-(3,4-dichloro-phenyl)-2,3-dihydro1H-isoindol-5-ylamino]-nicotinic acid (PD-307243) causes instantaneous
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