Structural implications of hERG K+ channel block by a high-affinity minimally structured blocker

Cardiac potassium channels encoded by human ether-à-go-go–related gene (hERG) are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK293 cells was inhibited with an IC50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold, respectively). The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block.

. A) Representative current traces of I hERG elicited by the step protocol illustrated in the lower panels in Control (i) and in the presence of 30 nM Cavalli-2 (ii); for clarity, selected currents at 4 test voltages are shown while the full protocol spanned from -40 to +60 mV in 10 mV increments. Peak I hERG tails at -40 mV following each depolarising command were measured relative to that elicited by the initial brief 50 ms step from -80 mV to -40 mV. B) Normalised I-V relationship for I hERG tails in Control (black) and in presence of 30 nM Cavalli-2 (grey). Peak tail currents in both conditions were normalized to the maximal tail current amplitude recorded in Control (n=5, & p<0.01 2-Way ANOVA with Bonferroni post hoc test). The experimental points were fitted with equation 3 of the main text. Control V 0.5 = -16.4 ± 0.8 mV, Slope 5.7 ± 0.6 and 30 nM Cavalli-2 V 0.5 = -21.1 ± 1.3 mV, Slope 4.8 ± 1.1. ΔV 0.5 = -4.68 mV, (n=5). A.
---------S5 ----------MthK 19: PATRILLLVLAVIIYGTAGFHFIEGESWT hERG 549: VLFLLMCTFALIAHWLACIWYAIEGESWT  Figure S2. A) Alignment of hERG and MthK S5 helix, pore helix, selectivity filter (SF; yellow highlight) and S6 helix sequences used to construct the MthK-based hERG pore homology model using PDB:1LNQ as a structural template. The MthK loop sequence (underlined) connecting the top of S5 and the pore helix was built into the hERG pore homology model to replace the turret region between S5 and the pore helix in hERG (see Figure 1 of main text). The structure of the pore from residues 613 to 663 in the hERG homology model is the same as the structure in the MthK-based hERG pore homology model described previously (Dempsey et al., 2014).
B) The hERG and MthK S5 sequences align poorly. The alignment was chosen which oriented F557 (red in the hERG sequence in A) towards the S6 helix at an equivalent height with respect to Y652 on S6 as in the hERG construct cryo-EM structure (Wang and MacKinnon, 2017), and which best reproduced the interactions between side chains at the top of S5 and in the pore helix as determined by distances (shown in B) between side chain β-carbons where these helices pack together in the cryo-EM structure.   Binding site (10 Å radius) centred above β-carbon of Y652 chain A. F656 side chains of chains B and D Cα-Cβ rotamer chosen to project side chains towards pore to allow multiple F656 interactions. 2 As above but free F656 rotamer sampling allowed.
A. Cavalli-2 docked into hERG cryo EM structure with side chain rotamers of F656 fixed to project side chains towards pore. Simplified stereo version of Figure 9b of main paper.
B. Cavalli-2 docked into hERG cryo EM structure with free rotation of F656 side chains. Simplified stereo version of Figure 9c of main paper.
C. Cavalli-2 docked into MthK-based hERG pore model with free rotation of F656 side chains. Simplified stereo version of Figure 10 of main paper. Figure S4. Stereo images of docking outputs described in the text.