Introduction of a single carboxylic acid converts the cyclic oligomeric depsipeptide ent-verticilide from a ryanodine receptor 2 (RyR2) inhibitor to RyR2 activator.

Cyclic oligomeric depsipeptides represent a distinct structural class of naturally occurring compounds known for their wide-ranging biological activities. We previously reported that the unnatural form of verticilide (ent-verticilide) inhibits cardiac ryanodine receptor 2 (RyR2) and exhibits antiarrhythmic effects in mice, but its mechanism of action on the RyR2 channel is not known. Here, we collected single-channel recordings in artificial lipid bilayers to elucidate the mechanism of RyR2 modulation by ent-verticilide and its polar side chain analog activert. ent-Verticilide reduced RyR2 activity by increasing the RyR2 mean closed time without changing the RyR2 mean open time, suggesting that ent-verticilide functions as a closed-channel stabilizer. ent-Verticilide exhibited partial inhibition on RyR2 single channels with an IC50 of ∼0.2 μM and a maximal inhibitory efficacy of ∼23%. To explore the effect of a charged residue on ent-verticilide-RyR2 binding, we introduced a terminal carboxylic acid on a single pentyl side chain. The resulting compound lost its inhibitory activity, increased RyR1 and RyR2 single-channel activity, and increased Ca spark frequency. Thus, we named this analog activert (1). Single-channel analysis showed that activert shortened mean closed time without changing mean open time, indicating closed-channel destabilization. Compared with ent-verticilide, activert was ∼100-fold less potent (EC50 ∼ 30 μM) on RyR2 and had low membrane permeability. RyR2 activation was confirmed by [3H]-ryanodine binding and Ca spark assays. Although poor membrane permeability represents an obstacle for therapeutic development, activert serves as a proof-of-concept partial RyR2 activator and a promising scaffold for future structure-activity optimization. SIGNIFICANCE STATEMENT: This study reveals the dual modulatory potential of cyclooligomeric depsipeptides on ryanodine receptor 2, with ent-verticilide acting as a closed-channel stabilizer and its analog, activert, functioning as a closed-channel destabilizer. By leveraging nonnatural enantiomers and rational scaffold modifications, we highlight an underexplored approach to uncover important structure-activity relationships, advancing the development of novel ryanodine receptor 2-targeted therapeutics with potential applications in cardiac arrhythmia management.