Stereoselective block of the hERG potassium channel by the Class Ia antiarrhythmic drug disopyramide

Abstract

Potassium channels encoded by human Ether-à-go-go-Related Gene (hERG) are inhibited by diverse cardiac and non-cardiac drugs. Disopyramide is a chiral Class Ia antiarrhythmic that inhibits hERG at clinical concentrations. This study evaluated effects of disopyramide enantiomers on hERG current (I_hERG) from hERG expressing HEK 293 cells at 37 °C. S(+) and R(-) disopyramide inhibited wild-type (WT) I_hERG with IC₅₀ values of 3.9 µM and 12.9 µM respectively. The attenuated-inactivation mutant N588K had little effect on the action of S(+) disopyramide but the IC₅₀ for the R(-) enantiomer was ~ 15-fold that for S(+) disopyramide. The enhanced inactivation mutant N588E only slightly increased the potency of R(-) disopyramide. S6 mutation Y652A reduced S(+) disopyramide potency more than that of R(-) disopyramide (respective IC₅₀ values ~ 49-fold and 11-fold their WT controls). The F656A mutation also exerted a stronger effect on S(+) than R(-) disopyramide, albeit with less IC₅₀ elevation. A WT-Y652A tandem dimer exhibited a sensitivity to the enantiomers that was intermediate between that of WT and Y652A, suggesting Y652 groups on adjacent subunits contribute to the binding. Moving the Y (normally at site 652) one residue in the N- terminal (up) direction in N588K hERG markedly increased the blocking potency of R(-) disopyramide. Molecular dynamics simulations using a hERG pore model produced different binding modes for S(+) and R(-) disopyramide consistent with the experimental observations. In conclusion, S(+) disopyramide interacts more strongly with S6 aromatic binding residues on hERG than does R(-) disopyramide, whilst optimal binding of the latter is more reliant on intact inactivation.

Keywords: Disopyramide; Enantiomer; HERG; Long QT; QT interval; Stereoselectivity.

Fig. 1

Effects of S(+) and R(−) Disopyramide on WT I_hERG. Representative I_hERG traces before (control) and after achieving steady-state block by S(+) disopyramide (A) and R(−) disopyramide (B) with the voltage protocol shown underneath. C shows concentration-response relation for S(+) and R(−) disopyramide (n = 5–7 for S(+) and n = 4–6 for R(−) disopyramide). D, E show rightwards shift of the concentration–response relation for S(+) disopyramide (D) and R(−) disopyramide (E) in 94 mM[K⁺]_e Tyrode’s solution(n = 5–7 for both S(+) and R(−) disopyramide) compared with 4 mM [K⁺]_e Tyrode’s

Fig. 2

Effects of mutant N588K and N588E on I_hERG inhibition by S(+) and R(−) Disopyramide. Representative traces of N588K I_hERG in the absence and presence of S(+) disopyramide (Ai) and R(−) disopyramide (Bi), elicited by the voltage protocol shown in the lower panel. Aii, Bii. Concentration–response relations for N588K I_hERG block compared with that for WT hERG for S(+) disopyramide (Aii, n = 4–7 for N588K) and R(−) disopyramide (Bii, n = 4–8 for N588K) or compared with each other (C). Di and Dii concentration–response relations for N588E I_hERG block compared with WT for S(+) disopyramide (Di, n = 5–8 for N588E) and R(−) disopyramide (Dii, n = 4–8 for N588E)

Fig. 3

Effects of S6 aromatic mutants Y652A and F656A on S(+) and R(−) disopyramide block of I_hERG. Representative traces of Y652A I_hERG in the absence and presence of S(+) disopyramide (Ai) and R(−) disopyramide (Bi), elicited by the voltage protocol shown in the lower panel. Aii, Bii. Concentration–response relations for Y652A I_hERG block compared with WT for S(+) disopyramide (Aii, n = 5–11 for Y652A) and R(−) disopyramide (Bii, n = 5–7 for Y652A). Representative traces of F656A I_hERG in the absence and presence of S(+) disopyramide (Ci) and R-disopyramide (Cii), elicited by the whole voltage protocol (left) or on expanded timescale (right), shown in the lower panels. Di, Dii. Concentration–response relations for F656A I_hERG block compared with WT for S(+) disopyramide (Di, n = 5–9 for F656A) and R(−) disopyramide (Dii, n = 5–8 for F656A).

Fig. 4

Effects of S6 tandem dimer D-Y652A (td [wt: Y652A]) on S(+) and R(−) disopyramide block of I_hERG. (Ai) Tandem dimers D-Y652A (td[wt:Y652A]) are shown as a concatenated molecule of the monomer WT(N-terminal) and monomer Y652A (C-terminal) with a short linker. (Aii) When functional channels incorporating dimers assemble, mutation sites should be located on subunits in the diagonal position of the tetrameric channel, WT subunits are indicated by ovals and the mutated subunits by rectangles. (Bi) Upper left inset panel shows the assembly of D-Y652A, oval represents WT monomer with residue Y in position 652, and rectangle represents Y652A mutant monomer with residue A in position 652. Lower left panel represents the possible contribution of residues to the binding on the subunits. Filled oval represent enantiomers interacting with residues on the subunits (solid lines indicate positive interactions, and dashed lines indicate possible interactions). Representative traces of tandem dimer Y652A I_hERG in the absence and presence of S(+) disopyramide (Bi) and R(−) disopyramide (Ci), elicited by the voltage protocol shown in the lower panel. (Bii, Cii). Concentration–response relations for WT, the tandem dimer Y652A I_hERG and homotetrameric Y652A I_hERG block by S(+) disopyramide (Bii, n = 5–11 for D-Y652A) and R-disopyramide (Cii, n = 5–7 for D-Y652A)

Fig. 5

Repositioning of aromatic residues on an N588K background. (A) Schematic showing sequences of S5-pore and S6 domain regions to show location of N588K and repositioning N588K-up or down mutants. (B-D) effects of repositioning S6 aromatic residue Y652 and F656 in attenuated inactivation N588K channels on I_hERG blocking potency of R(−) disopyramide. Representative traces of N588K-Y-up I_hERG in the absence and presence of R(-) disopyramide (Bi, n = 5–8 for N588K-Y-up), elicited by the voltage protocol shown in the lower panel and its concentration-response relation (Bii), Representative traces of N588K-Y-down I_hERG in the absence and presence of R(−) disopyramide (Ci, n = 5–6 for N588K-Y-down), elicited by the voltage protocol shown in the lower panel and its concentration-response relation (Cii). Representative traces of N588K-F-up I_hERG in the absence and presence of R(−) disopyramide (Di, n = 5–8 for N588K-F-up), elicited by the voltage protocol shown in the lower panel and its concentration–response plot (Dii)

Fig. 6

Stereoselective interactions of disopyramide enantiomers within a hERG pore homology model. Each panel is a snapshot of the interaction of a disopyramide enantiomer obtained during 200 ns MD runs within a fully hydrated, membrane-incorporated MthK-based hERG pore homology model (see Supplementary Figs. 6 and 7 for the structure of the membrane system and starting structures of each disopyramide enantiomer within the pore cavity). Disopyramide enantiomers are represented as yellow sticks and hERG pore amino acid side chains are Y652 (pink), F656 (blue) and S624 (green). Discrete drug-pore interactions as defined in [44] are shown as black (aromatic stacking), blue (cation-pi) and green (hydrogen bond) dotted lines. The location of the tertiary aliphatic amino group in or near the binding site for a hydrated K⁺ ion below the selectivity filter is indicated with a blue star. The purple sphere is a K⁺ ion in the 4-position of the selectivity filter. The Y652 side chains that interact with disopyramide lie on adjacent pore subunits in both S(+) and (R−) bound structures. Stereo views of these binding modes are shown in Supplementary Figs. 8 and 9