Pharmacological and electrophysiological characterization of AZSMO-23, an activator of the hERG K(+) channel

Abstract

Background and purpose: We aimed to characterize the pharmacology and electrophysiology of N-[3-(1H-benzimidazol-2-yl)-4-chloro-phenyl]pyridine-3-carboxamide (AZSMO-23), an activator of the human ether-a-go-go-related gene (hERG)-encoded K(+) channel (Kv 11.1).

Experimental approach: Automated electrophysiology was used to study the pharmacology of AZSMO-23 on wild-type (WT), Y652A, F656T or G628C/S631C hERG, and on other cardiac ion channels. Its mechanism of action was characterized with conventional electrophysiology.

Key results: AZSMO-23 activated WT hERG pre-pulse and tail current with EC50 values of 28.6 and 11.2 μM respectively. At 100 μM, pre-pulse current at +40 mV was increased by 952 ± 41% and tail current at -30 mV by 238 ± 13% compared with vehicle values. The primary mechanism for this effect was a 74.5 mV depolarizing shift in the voltage dependence of inactivation, without any shift in the voltage dependence of activation. Structure-activity relationships for this effect were remarkably subtle, with close analogues of AZSMO-23 acting as hERG inhibitors. AZSMO-23 blocked the mutant channel, hERG Y652A, but against another mutant channel, hERG F656T, its activator activity was enhanced. It inhibited activity of the G628C/S631C non-inactivating hERG mutant channel. AZSMO-23 was not hERG selective, as it blocked hKv 4.3-hKChIP2.2, hCav 3.2 and hKv 1.5 and activated hCav 1.2/β2/α2δ channels.

Conclusion and implications: The activity of AZSMO-23 and those of its close analogues suggest these compounds may be of value to elucidate the mechanism of type 2 hERG activators to better understand the pharmacology of this area from both a safety perspective and in relation to treatment of congenital long QT syndrome.

Figure 1

Chemical structure of AZSMO-23: (N-[3-(1H-benzimidazol-2-yl)-4-chloro-phenyl]pyridine-3-carboxamide).

Figure 2

Activation of hERG current by AZSMO-23. (A) Typical leak-corrected current trace from IonWorks HT before and 3 min after exposure to 100 μM AZSMO-23. The dashed line represents the zero current level after leak subtraction. (B) Concentration–effect relationship for AZSMO-23 versus the pre-pulse current and tail current. Current amplitudes in the presence of a given concentration of AZSMO-23 were normalized by expressing them as a percentage of the mean, time-matched vehicle control data collected in the same PatchPlate. The dashed line at 100% therefore represents the vehicle control level for the pre-pulse or tail current. Each data point is mean ± SEM; n = 14–20. For most data points, the error bar is covered by the symbol. The concentration–effect curve parameters for the pre-pulse data are: EC₅₀ 28.6 μM [lower and upper 95% confidence limit (CL) 25.9, 32.6 μM respectively]; n_H 2.2. The corresponding tail current data are: EC₅₀ 11.2 μM (lower and upper 95% CL 6.8, 18.4 μM respectively); n_H 3.6.

Figure 3

AZSMO-23 time course of effect. Using data from IonWorks Barracuda, (A) shows the onset of, and recovery from, the effect of AZSMO-23 (30 μM) on pre-pulse current compared with the effects of vehicle. (B) As for A, but measuring the effect on tail current compared with vehicle. Current amplitudes are expressed relative to the pre-pulse or tail current amplitude evoked at 150 s (i.e. immediately before addition of AZSMO-23 or vehicle). Each data point is mean ± SEM; n = 8.

Figure 4

Effect of 30 μM AZSMO-23 on hERG pre-pulse and tail current activation. All data are from conventional whole-cell electrophysiology. (A) The voltage protocol used (top) to evoke typical current responses after 5 min exposure to vehicle (middle) or AZSMO-23 (bottom). Note that only the current traces to V_pre-pulse steps of −40, −30, −20, −10 and 0 mV are shown. The dashed line represents the zero current level. (B) Pre-pulse current–voltage relationship in cells after 5 min exposure to vehicle or AZSMO-23. (C) Tail current–voltage relationship in cells after 5 min exposure to vehicle or AZSMO-23. (D) Normalized data from C. For the graphs in B, C and D, each data point is mean ± SEM; n = 6 (vehicle cells); n = 7 (AZSMO-23 cells).

Figure 5

Effect of 30 μM AZSMO-23 on hERG voltage dependence of inactivation. All data are from conventional whole-cell electrophysiology. (A) The voltage protocol used (top) to evoke typical current responses after 5 min exposure to vehicle (middle) or AZSMO-23 (bottom). Note that only the current traces to V_pre-pulse steps of +80, +40, 0, −40, −80 and −120 mV are shown. Furthermore, only the initial current trace after stepping from −80 to +40 mV, and the 20 ms of current data preceding the V_pre-pulse onwards are shown. The dashed line represents the zero current level. (B) The current–voltage relationship after 5 min exposure to vehicle or AZSMO-23. (C) Normalized data from B. Each data point is mean ± SEM; n = 6 (vehicle cells); n = 7 (AZSMO-23 cells).

Figure 6

Effect of AZSMO-23 on a non-inactivating hERG mutant channel (G628C/S631C). Typical leak-corrected current trace, using IonWorks HT, before and after 100 μM AZSMO-23. The dashed line represents the zero current level after leak subtraction.

Figure 7

Effect of AZSMO-23 and its close analogues on hERG tail current. Concentration–effect relationship for AZSMO-23 and its close analogues, compounds 1, 2 and 3, using IonWorks HT. Current amplitudes in the presence of a given concentration of test compound were normalized by expressing them as a percentage of the mean, time-matched, vehicle control data collected in the same PatchPlate. The dashed line at 100% therefore represents the tail current vehicle control level. Each data point is mean ± SEM; n = 4–11. AZSMO-23 concentration–effect curve parameters for the tail current data shown are: EC₅₀ 12.3 μM (lower and upper 95% CL 8.7, 20.1 μM respectively); n_H 3.4. Concentration–effect curve parameters were not defined for AZSMO-23 analogues since curves were incomplete.

Figure 8

Effect of combining structural features of AZSMO-23 and ICA-105574. (A) Structures of AZSMO-23, ICA-105574 and the N-phenyl benzamide core common to both compounds. The ICA-105574 structural element is shown in blue and the AZSMO-23 structural element shown in red, were exchanged to make compounds 4 and 5. (B) Concentration–effect relationship for compounds 4 and 5, using IonWorks HT. Current amplitudes in the presence of a given concentration of test compound were normalized by expressing them as a percentage of the mean, time-matched, vehicle control data collected in the same PatchPlate. The dashed line at 100% therefore represents the tail current vehicle control level. Each data point is mean ± SEM; n = 4–8. Concentration–effect curve parameters were not defined since curves were incomplete.

Figure 9

Effect of cisapride or AZSMO-23 on Y652A and F656T hERG mutant channels. Data are from experiments using IonWorks HT. Concentration–effect curves shown are for pre-pulse current (A, C, E, G) and for tail current (B, D, F, H). The top four graphs are for cisapride (A, B, C, D) and the bottom four (E, F, G, H) are for AZSMO-23. Current amplitudes in the presence of a given concentration of test compound were normalized by expressing them as a percentage of the mean, time-matched, vehicle control data collected in the same PatchPlate. The dashed line at 100% therefore represents the pre-pulse or tail current vehicle control level. Each data point is mean ± SEM (see Table 1 for n numbers). Concentration–effect curve parameters are summarized in Table 1.

Figure 10

Typical current traces showing the effect of AZSMO-23 on Y652A and F656T hERG mutant channels. (A) Typical leak-corrected current traces, using IonWorks HT, for Y652A hERG channels before and after 30 μM AZSMO-23. (B) Equivalent current traces for F656T hERG. The dashed line represents the zero current level after leak subtraction.

Figure 11

Effect of AZSMO-23 on other cardiac ion channel types. Concentration–effect curves for AZSMO-23 on hCa_v3.2 and hK_v1.5 channels using IonWorks HT, and hK_v4.3-hKChIP2.2 and hCa_v1.2/β2/α2δ channels, using IonWorks Quattro. Current amplitudes in the presence of a given concentration of test compound were normalized by expressing them as a percentage of the mean, time-matched, vehicle control data collected in the same PatchPlate. The dashed line at 100% therefore represents the vehicle control level. Each data point is mean ± SEM. n values were: hCa_v3.2 (4–8 cells); hK_v1.5 (7–8 cells); hK_v4.3-hKChIP2.2 (10–16 wells); hCa_v1.2/β2/α2δ (6–8 wells). Concentration–effect curve parameters were not defined since curves were incomplete.