Unnatural verticilide enantiomer inhibits type 2 ryanodine receptor-mediated calcium leak and is antiarrhythmic

Ca ²⁺ leak via ryanodine receptor type 2 (RyR2) can cause potentially fatal arrhythmias in a variety of heart diseases and has also been implicated in neurodegenerative and seizure disorders, making RyR2 an attractive therapeutic target for drug development. Here we synthesized and investigated the fungal natural product and known insect RyR antagonist (−)-verticilide and several congeners to determine their activity against mammalian RyR2. Although the cyclooligomeric depsipeptide natural product (−)-verticilide had no effect, its nonnatural enantiomer [ent-(+)-verticilide] significantly reduced RyR2-mediated spontaneous Ca ²⁺ leak both in cardiomyocytes from wild-type mouse and from a gene-targeted mouse model of Ca ²⁺ leak-induced arrhythmias (Casq2 ⁻ / ⁻ ). ent-(+)-verticilide selectively inhibited RyR2-mediated Ca ²⁺ leak and exhibited higher potency and a distinct mechanism of action compared with the pan-RyR inhibitors dantrolene and tetracaine and the antiarrhythmic drug flecainide. ent-(+)-verticilide prevented arrhythmogenic membrane depolarizations in cardiomyocytes without significant effects on the cardiac action potential and attenuated ventricular arrhythmia in catecholamine-challenged Casq2 ⁻ / ⁻ mice. These findings indicate that ent-(+)-verticilide is a potent and selective inhibitor of RyR2-mediated diastolic Ca ²⁺ leak, making it a molecular tool to investigate the therapeutic potential of targeting RyR2 hyperactivity in heart and brain pathologies. The enantiomer-specific activity and straightforward chemical synthesis of (unnatural) ent-(+)-verticilide provides a compelling argument to prioritize ent-natural product synthesis. Despite their general absence in nature, the enantiomers of natural products may harbor unprecedented activity, thereby leading to new scaffolds for probe and therapeutic development.