Dual Modulation of Cardiac Ion Pumps: A Small-Molecule SERCA2a SUMOylation Enhancer Also Inhibits the Na⁺/K⁺-ATPase

Background: The Na⁺/K⁺-ATPase (NKA) maintains electrochemical gradients by exporting Na+ and importing K⁺ at the expense of ATP hydrolysis. Although NKA inhibition is a well-established strategy for increasing cardiac contractility, existing inhibitors such as cardiotonic steroids (CTS) are limited by serious adverse effects. N106 is a small molecule previously shown to enhance cardiac lusitropy by promoting SERCA2a SUMOylation and, intriguingly, also exerts positive inotropic effects, suggesting additional mechanisms of action. Methods: To test whether N106 directly modulates NKA, we combined ATPase activity assays with molecular docking and microsecond-scale molecular dynamics simulations. Results: Biochemical measurements showed that N106 partially inhibits NKA, achieving ~80% maximal inhibition with an IC50 of 7 ± 1 µM, while leaving the pump's apparent affinity for Na⁺, K⁺, and ATP unchanged. Computational analyses suggest that N106 binds within the canonical CTS-binding pocket but undergoes intermittent unbinding events, consistent with the partial inhibition observed experimentally. Conclusions: These findings identify N106 as a first-in-class dual modulator of cardiac ion pumps, partially inhibiting NKA while previously shown to activate SERCA2a through enhanced SUMOylation. This combined mechanism likely underlies its positive inotropic and lusitropic effects and positions the N106 scaffold as a promising lead for developing next-generation dual-target therapeutics for heart failure.