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

Cardiac potassium channels encoded byhuman 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 HEK 293 cells was inhibited with an IC50of 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, whilst 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 F656 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 F656 side chain orientations compatible with high affinity block.