PROJECT SUMMARY Ryanodine Receptor 2 (RyR2) mutations that result in dysregulated RyR2-mediated calcium release are associated with a variety of cardiovascular diseases. As a result, several small molecule probes have been used in the study and treatment of cardiac diseases through the targeting of RyR calcium channels. Flecainide, tetracaine, and dantrolene, the most common of these small molecule probes, are not without problems. Flecainide, for example, is contra-indicated in patients with structural heart defects or heart failure. Furthermore, selectivity of specific RyR isoforms (RyR1, RyR2, or RyR3) is rare, and to date, an RyR2-selective probe does not exist. New probes to selectively modulate RyR2 would be of great use both therapeutically and as tools to better understand the function of RyR2 and the mechanism of intracellular calcium flux. This proposal focuses on the development of a new class of antiarrhythmic agents using the hit compound discovered in the Johnston lab, ent-verticilide – the non-natural enantiomer of the natural product (–)-verticilide. This cyclic oligomeric depsipeptide is a potent and selective inhibitor of RyR2-mediated calcium release in mammals while the natural verticilide is inactive. We aim to use a structure-based design approach to help clarify the interaction of ent- verticilide with RyR2. This approach may lead to new findings in our search for potent, selective modulators of RyR2 for the treatment of cardiac disease. Our screening will be grounded first in the well-defined RyR2-mediated spontaneous calcium release assay. We aim to include both discrete collections of ring-size congeners and ‘unnatural’ enantiomers, two underexplored variables in natural-product based therapeutic development, to screen against the target (RyR2), potentially leading to new probes. We also propose to use ent-verticilide in pharmacological studies to determine ADME, while advancing the understanding of the mechanism of action and RyR2 function. Our approach is well suited towards the ultimate goals of both developing a potent, selective inhibitor of RyR2 as well as working towards defining the mechanism of action of ent-verticilide to treat cardiac arrythmias.