Novel strong determinant of hERG channel inhibition on segment S5 (F557)

P SAXENA, T LINDER, E TIMIN, S HERING, A STARY-WEINZINGER. Depatment of Pharmacology & Toxicology, University of Vienna, Vienna, Austria

The human ether-a-go-go related gene (hERG, Kv11.1) plays a pivotal role in repolarization of the ventricles. Understanding the molecular basis of the arrhythmogenicity of hERG inhibition is, therefore, essential for drug development. We have previously shown that pi-pi stacking interactions between the two cavity facing aromatic amino acids Y652 and F656 on S6 helix can shape the hERG drug binding pocket. Loss of pi-pi stacking interactions caused by Y652A may induce conformational changes in the F656 side chain and affect the binding orientation of blockers.[1]

MD simulations and docking revealed that an aromatic amino acid (F557) located in close proximity on S5 helix may also influence the conformation of the drug binding residues (Y652/F656) and thereby directly modulate channel inhibition.

To test this hypothesis, we investigated the effect of the F557L on hERG inhibition by four different hERG blockers. hERG channels were heterologously expressed in Xenopus laevis oocytes and channel inhibition studied by means of the two-microelectrode voltage clamp technique. Comparable IC50 shifts towards higher drug concentrations were induced by F557L and Y652A for all four compounds (e.g. haloperidol: ΔIC50 (F557L/Y652A) = 43.6/49.3 µM. Remarkably, F557L completely prohibits channel block by dofetilide.

Our data may be interpreted in two ways: First, the IC50 shifts in F557L are caused by the loss of pi-pi stacking interactions and resulting changes in the orientation of the drug binding determinants Y652 and F656. Second, drugs may interact with F557 directly. Docking studies suggest a binding mode orthogonal to the channel between two adjacent subunits supporting the second hypothesis.

Acknowledgements:

This work was supported by the Austrian Science Fund (FWF; Grants P22395). T.L. was supported by a research fellowship 2013 from the University of Vienna. P.S. and T.L. are fellows of the graduate school program MolTag (FWF W1232).

1. Knape K, Linder T, Wolschann P, Beyer A, Stary-Weinzinger A (2011) In silico Analysis of Conformational Changes Induced by Mutation of Aromatic Binding Residues: Consequences for Drug Binding in the hERG K+ Channel. PloS one 6: e28778. doi:10.1371/journal.pone.0028778.