Ca2+ leak from cardiac ryanodine receptor (RyR2) is an established mechanism of sudden cardiac death (SCD), whereby dysregulated Ca2+ handling causes ventricular arrhythmias. We previously discovered the RyR2-selective inhibitor ent-(+)-verticilide (ent-1), a 24-membered cyclooligomeric depsipeptide that is the enantiomeric form of a natural product (nat-(-)-verticilide). Here, we examined its 18-membered ring-size oligomer (ent-verticilide B1; ent-B1) in single RyR2 channel assays, [3H]ryanodine binding assays, and in Casq2-/- cardiomyocytes and mice, a gene-targeted model of SCD. ent-B1 inhibited RyR2 single-channels and [3H]ryanodine binding with low micromolar potency, and RyR2-mediated spontaneous Ca2+ release in Casq2-/- cardiomyocytes with sub-micromolar potency. ent-B1 was a partial RyR2 inhibitor, with maximal inhibitory efficacy of less than 50%. ent-B1 was stable in plasma, with a peak plasma concentration of 1460 ng/ml at 10 min and half-life of 45 min after intraperitoneal administration of 3 mg/kg in mice. Both 3 mg/kg and 30 mg/kg ent-B1 significantly reduced catecholamine-induced ventricular arrhythmia in Casq2-/- mice. Hence, we have identified a novel chemical entity, ent-B1, that preserves the mechanism of action of a hit compound and shows therapeutic efficacy. These findings strengthen RyR2 as an antiarrhythmic drug target and highlight the potential of investigating the mirror-image isomers of natural products to discover new therapeutics.
The unnatural verticilide enantiomer (ent-verticilide) is a selective and potent inhibitor of cardiac ryanodine receptor (RyR2) calcium release channels and exhibits antiarrhythmic activity in a murine model of catecholaminergic polymorphic ventricular tachycardia (CPVT). To determine verticilide's pharmacokinetic and pharmacodynamic properties in vivo, we developed a bioassay to measure nat-and ent-verticilide in murine plasma and correlated plasma concentrations with antiarrhythmic efficacy in a mouse model of CPVT. nat-Verticilide rapidly degraded in plasma in vitro, showing >95% degradation within five minutes, whereas ent-verticilide showed <1% degradation over six hours. Plasma was collected from mice following intraperitoneal administration of ent-verticilide at two doses (3 mg/kg, 30 mg/kg). Peak plasma concentration (C max ) and area under the plasma-concentration time curve (AUC) scaled proportionally to dose and the half-life was 6.9 hr for the 3 mg/kg dose and 6.4 hr for the 30 mg/kg dose.Antiarrhythmic efficacy was examined using a catecholamine challenge protocol at time points ranging from 5 to 1440 min after intraperitoneal dosing. ent-Verticilide inhibited ventricular arrhythmias as early as 7 min after administration in a concentration-dependent manner, with an estimated potency (IC 50 ) of 266 ng/mL (312 nM) and an estimated maximum inhibitory effect of 93.5%. Unlike the FDA-approved pan-RyR blocker dantrolene, the RyR2-selective blocker entverticilide (30 mg/kg) did not reduce skeletal muscle strength in vivo. We conclude that entverticilide has favorable pharmacokinetic properties and reduces ventricular arrhythmias with an estimated potency in the nanomolar range, warranting further drug development.
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