hERG MAP-A rational approach to eliminate ion channel related cardiotoxicity

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DESCRIPTION (provided by applicant): hERG K+ channel inhibition by drugs is implicated in cardiac arrhythmias and sudden death. Several drugs were removed from the market due to hERG-related cardiotoxicity New methods are needed for eliminating hERG binding to new drugs. Drug development will benefit substantially from new experimentally-derived insights into the structural basis for hERG block. Neurion has developed a unique approach, the hERG MAP(tm) that identifies the key interactions drugs make with the hERG channel. The data guide medicinal chemists in synthetically reducing or eliminating hERG binding. We validated our rational approach in successful collaborations with major pharmaceutical companies. Currently we express hERG channels in Xenopus oocytes. The yolk of the oocyte is large and hydrophobic, and appears to adsorb drugs. Importantly, yolk binding makes drug affinities estimates measured in oocytes appear less than those measured in mammalian cells. This limits our ability to directly estimate affinity and to measure weakly binding drugs. In order make our current approach more widely applicable and in line with current pharmaceutical company practice, we propose to adopt hERG MAP for mammalian cell expression systems. In Aim 1 we will elucidate the binding interactions and channel block of six drugs with WT hERG and hERG mutated at critical binding residues expressed in mammalian cells; in Aim 2 we will apply the in vivo nonsense suppression methodology to generate unnatural hERG mutants using laser based (optical) injection to deliver exogenous amino acylated tRNA into mammalian cells. The result of the Phase I work will be wildly applicable, mammalian cell-based hERG assays that can directly aid medicinal chemists in eliminating hERG toxicity. In Phase II we will industrialize the assay by establishing the expression of all relevant hERG mutants in mammalian cells and optimizing the technology for higher- throughput, automated electrophysiology measurements. We will also assay a substantial number of molecules and conduct the computational interpretation needed to create a large database of drug-hERG interactions. Finally, we will also demonstrate the synthetic "rescue" of a known hERG-blocking molecule.