Structural requirements for high-affinity block of hERG K⁺ channels using electrophysiology and mutagenesis with simplified blockers

  • Matthew Helliwell

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)


Human ether-à-go-go-related gene (hERG) potassium channels, which conduct the rapid delayed rectifier current in cardiomyocytes, are of great medical and pharmaceutical relevance as hERG inhibition by structurally and therapeutically diverse drugs is associated with acquired long QT syndrome (aLQTS) and an increased risk of sudden death. Cavalli-2 is a highly potent ‘minimally-structured’ hERG-blocking compound synthesised in an attempt to ascertain the minimal structural requirements for high-affinity hERG block (Cavalli 2012). The aim of the study was to use minimally-structured (Cavalli-2) and simplified hERG blockers (E-4031-17 and its parent E-4031) with electrophysiology and mutagenesis to define the structural determinants of hERG block within the drug binding site.
A detailed analysis of Cavalli-2 showed it acted as a gated-state dependent hERG blocker and its inhibitory action was contingent on intact inactivation although the molecule bound slightly more strongly to the open state. The concentration-dependence of block by Cavalli-2 allowed accurate IC50 values to be determined for 7 mutants. Mutagenesis of the pore region highlighted that Cavalli-2 likely forms multiple interactions with Phenylalanine-656 side chains. Ala-mutation of the pore-helix residue Serine-624 attenuated IhERG block by E-4031-17, indicating that S624 side chains play a role in high affinity block other than as hydrogen bond donors. Mutation of the S5 aromatic residue Phenylalanine-557L attenuated IhERG block by all compounds studied and abolished the voltage-dependence of block by Cavalli-2, implicating F557 as an important binding determinant.
Computational docking of all drugs into one of the hydrophobic pockets identified in a recently published Cryogenic Electron Microscopy (Cryo-EM) hERG structure supported direct interactions involving F557 side chains; however docking outputs were not found where drugs could interact with multiple F656 side chains despite the profound attenuation of block observed by all three drugs in F656A channels. The poor correspondence between Alanine-scanning mutagenesis data and computational docking with the cryo-EM structure of hERG suggests that the cryo-EM may have captured hERG in a conformation that is poorly compatible for high-affinity drug block.
Date of Award7 May 2019
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorJules C Hancox (Supervisor) & Christopher E Dempsey (Supervisor)

Cite this

Structural requirements for high-affinity block of hERG K⁺ channels using electrophysiology and mutagenesis with simplified blockers
Helliwell, M. (Author). 7 May 2019

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)