Using Unnatural Amino Acids To Probe The Molecular Basis For Herg Drug Block

The human Ether-a-go-go Related Gene (hERG) voltage gated potassium channel underlies the rapid delayed rectifier current, IKr, which plays a central role in cardiac action potential repolarization. Inhibition of IKr results in a heart rhythm disorder, long QT syndrome type 2, which can lead to torsades de pointes and sudden death. Unfortunately, hERG is blocked by a wide range of therapeutic drug classes and this has led federal drug regulators to mandate hERG-block screening as a requirement in drug development. Past mutagenesis studies have highlighted two aromatic residues, Y652 and F656, in the pore region of hERG as crucial for high affinity drug block of the channel [1]. The molecular basis for drug interaction with these sites is often suggested to be via cation-pi interactions. We have directly tested this hypothesis using unnatural amino acid mutagenesis to incorporate fluorinated phenylalanine derivatives at Y652 and F656 in Xenopus laevis oocytes. Unnatural side chains can be efficiently incorporated into hERG at both positions tested, with very little non-specific incorporation. Using two-electrode voltage clamp recordings, we measured the affinity of known hERG blockers terfenadine and quinidine on WT and fluoro-Phe substituted channels. The effects of fluorination are not consistent with a cation-pi interaction as the primary force governing the action of these drugs. However, our findings demonstrate the feasibility of the nonsense suppression approach in hERG, and pave the way for detailed dissection of the chemical basis of hERG blockade.1. Mitcheson, J.S., et al., A structural basis for drug-induced long QT syndrome. Proc Natl Acad Sci U S A, 2000. 97(22): p. 12329-33.