Understanding the molecular basis of hERG (Kv11.1) inhibition is essential for drug development. Here we systematically analyzed the mechanism and potency of hERG inhibition by a library of 13 dofetilide derivatives1. Currents through hERG channels stably expressed in HEK 293 cells were studied applying whole-cell configuration using planar patch clamp technique2.The estimated IC50s ranged between 4.3±0.7nM (LUF6200) and 297±77nM (LUF6139). An inverse relation was observed between potency (IC50) and rate of block development. Analysis of the kinetics of block development revealed that potency of dofetilide derivatives is predominantly determined by the drug dissociation rate constant. In general, higher molecular weight led to higher apparent drug affinity (r = 0.9).LUF6200, the most active compound, has 2 nitro groups in para position of the two aromatic rings and an ethyl moiety at the basic nitrogen atom. Pursuing a Topliss approach at the phenoxy group further supports the positive contribution of the nitro group1, showing that substituents with electron withdrawing groups in para position of the phenyl ring are beneficial for hERG blocking activity. This points towards a pi-pi interaction of at least one of the aromatic rings of dofetilide analogs with respective aromatic amino acids in the channel. Docking of the set of compounds into a homology model of the hERG channel supports this finding. A refined molecular model for dofetilide-hERG interaction based on the novel functional data and mutational studies is proposed.1. Shagufta et al. Exploring Chemical Substructures Essential for hERG K+ Channel Blockade by Synthesis and Biological Evaluation of Dofetilide Analogues. ChemMedChem 4, 1722-1732 (2009).2. Farre, C. & Fertig, N. HTS techniques for patch clamp-based ion channel screening - advances and economy. Expert Opin. Drug Discov. 7, 515-524 (2012).