An in Vitro Assay of hERG K+ Channel Potency for a New EGFR Inhibitor FHND004

Figures

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  FIGURE 1. Effects of FHND004 on IhERG recorded from HEK293 cells. (A) Families of IhERG elicited by 2 s pulses to the potentials between –70 and +60 mV in 10 mV increments from a holding potential of –80 mV and then back to –40 mV for 4 s in the absence and presence of 10 μM FHND004. (B) I–V relationships for tail current in the absence and presence of 10 μM FHND004 (n = 8). (C) Steady-state activation curve fitted by a Boltzmann equation in the absence and presence of 10 μM FHND004, respectively. (D) Time course of FHND004-induced inhibition of hERG tail current from the same cells (n = 5). (E) The summarized peak current density in the absence and presence of 10 μM FHND004. (F) Peak current elicited by a 30 mV depolarization pulse for 2 s and then back to –40 mV for 4 s under various concentrations of AZD9291 and FHND004, respectively. (G) Concentration–response relationships fitted with logistic equation for block of hERG tail current by AZD9291 and FHND004, respectively (n ≥ 6 per data point). And the slope factor (k) for AZD9291 is 4.70 and k for FHND004 is 3.50. Panel (G) adapted from Zhang et al. (2017) with permission.

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  FIGURE 2. Effects of FHND004 on IKr recorded from HL-1 cells. (A) Families of hERG current elicited by the voltage protocol shown in the inset. (B) Peak current elicited by the voltage protocol shown in the inset in the absence and presence of 10 μM FHND004. (C,D) I–V relationships for step and tail currents in the absence and presence of 10 μM FHND004, respectively (n = 6). (E) Concentration–response relationships fitted with Logistic equation for block of Ikr tail current by FHND004 (n ≥ 4 per data point); k is 2.21 ± 0.67.

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  FIGURE 3. Open channel block of hERG by FHND004. (A) Representative current elicited by the “envelope of tails” protocol before and after application of 10 μM FHND004. Cells were held at a holding potential of -80 mV and pulsed to depolarizing voltage (+30 mV) for variable durations from 20 to 1420 ms in 100 ms increments. Envelope tail currents were recorded upon repolarization to -40 mV. (B) Mean plots of current density against development of depolarizing time fitted with single exponential equation before and after the application of 10 μM FHND004 (n = 6). (C) A plot of the onset of current block expressed as relative tail current [relative tail current = (currents with drug/currents without drug) × 100%]. The time-dependent decay in relative tail current was fitted to a mono-exponential equation (n = 6). (D) Representative current traces recorded before and after the addition of 10 μM FHND004, using the protocol shown in the inset (n = 5).

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  FIGURE 4. Effects of FHND004 on hERG channel kinetics. (A) Representative current traces of steady-state inactivation before and after the application of 10 μM FHND004 (left) with the protocol shown in the inset and I–V relationships of the outward inactivation current elicited during the second step to +20 mV obtained in the absence and presence of 10 μM FHND004 (right, n = 6). (B) Representative current traces of the time-course of hERG inactivation recorded by a three-pulse protocol before and after the application of 10 μM FHND004 (left) and inactivation time constant (tau) fitted by a single exponential equation (right, n = 5). (C) Representative current traces of recovery from inactivation before and after application of 10 μM FHND004 (left) and the time constant (tau) of recovery from the inactivation fitted by a single exponential equation (right, n = 6). (D) Representative current traces of deactivation before and after the application of 10 μM FHND004 (left) and the fast and slow time constants (tau) fitted by a double exponential equation (right, n = 7).

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  FIGURE 5. Effects of A422T and H562P mutations on FHND004-induced inhibition. (A) Representative current traces recorded from HEK293 cells expressing WT-hERG, A422T-hERG, and H562P-hERG K+ channels, respectively. (B) Representative current traces recorded from HEK293 cells expressing WT/A422T- and WT/H562P-hERG K+ channels in the absence of 10 μM FHND004. (C) Representative current traces recorded from HEK293 cells expressing WT/A422T- and WT/H562P-hERG K+ channels in the presence of 10 μM FHND004 and concentration–response relationships fitted with logistic equation for block of WT-, WT/A422T-, and WT/H562P-hERG tail currents by FHND004 with the same voltage protocol shown in Figure 1E (n ≥ 6 per data point), and the slope factors are 3.50, 3.45, and 2.55, respectively.

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  FIGURE 6. Molecular determinants of hERG channel block by FHND004. (A) Current traces of WT-hERG and F656C in high [K+]e (94 mM) bath solution before and after the application of FHND004 with the protocol used to elicit the inward tail current shown in the inset and concentration–response relationships fitted with logistic equation for block of WT- and F656C-hERG inward tail current by FHND004 (n ≥ 5 per data point), and the slope factors are 3.00 and 3.61, respectively. (B) Current traces of WT-hERG, and Y652A, S624A, and F557L mutations before and after the application of FHND004 and concentration–response relationships fitted with logistic equation for block of WT- and Y652A-, S624A-, and F557L-hERG tail currents by FHND004 (n ≥ 6 per data point), and the slope factors are 3.50 ± 6.67, 3.00 ± 1.87, 2.86 ± 1.63, and 3.00 ± 1.39, respectively.