Identification of a Small Secretoneurin Derivative That Inhibits <scp>CaMKIIδ</scp> Activity

Ventricular arrhythmias, a major cause of sudden cardiac death, are driven by Ca²⁺ imbalance in cardiac myocytes, often linked to the overactivation of CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase II delta). As such, inhibiting CaMKIIδ represents a promising therapeutic strategy. Based on our previous finding that native secretoneurin (SN) is a weak CaMKIIδ inhibitor, we aimed to develop a more potent derivative of SN to effectively counter aberrant Ca²⁺ handling and arrhythmia risk. Various regions of SN were tested for CaMKII binding, identifying the core region as the sequence with the strongest binding capacity. This region was subsequently optimised with two phenylalanine substitutions, resulting in the SN derivative SN-db-short. Structural homology modeling and ELISA-based assays revealed that SN-db-short bound both the substrate-binding (S-site) region of CaMKIIδ, in addition to the ATP-binding region, with 8-fold stronger binding compared to SN. Surface plasmon resonance experiments confirmed that SN-db-short exhibited a higher association rate and affinity for CaMKIIδ compared to SN. Consistent with only a partial calmodulin binding motif, SN-db-short showed no calmodulin binding, indicating selective CaMKIIδ inhibition. In functional studies, SN-db-short inhibited CaMKIIδ-mediated phosphorylation of ryanodine receptor 2 and appeared more effective than SN in reducing the incidence of Ca²⁺ sparks and Ca²⁺ waves. SN-db-short also more markedly inhibited CaMKIIδ phosphorylation of phospholamban, slowed Ca²⁺ reuptake, and reduced the magnitude of Ca²⁺ transients during isoproterenol stimulation. SN-db-short effectively inhibits CaMKIIδ and significantly counters aberrant Ca²⁺ handling in cardiomyocytes. Thus, this optimised peptide holds therapeutic potential for reducing the risk of ventricular arrhythmias.