Discovery of Ro 41-0960, a Non-Steroidal Inhibitor of the Na⁺/K⁺-ATPase

The Na⁺/K⁺-ATPase (NKA) is a central regulator of cardiac ion homeostasis and a validated target for heart failure. Yet, cardiotonic steroids used in clinical practice are limited by a narrow therapeutic window and pro-arrhythmic effects. Here, we identify Ro 41-0960 as a non-steroidal inhibitor of NKA with a distinct mechanistic profile. The compound inhibits NKA activity with IC₅₀ values of 17.9 ± 1.1 µM for purified enzyme and 10.3 ± 1.1 µM in microsomal preparations. ATP-dependent activity measurements revealed a non-monotonic response in which inhibition was most pronounced at ATP concentrations below 4 mM and diminished at the highest ATP concentrations tested. Docking and molecular dynamics simulations suggest that Ro 41-0960 can access both the cardiotonic steroid-binding pocket and the nucleotide-binding site; however, the ATP-dependence data are not consistent with a simple ATP-competitive mechanism. Furthermore, the compound shows only weak inhibition of SERCA (IC₅₀ > 100 µM) and does not alter electrophysiological parameters in human iPSC-derived cardiomyocytes at concentrations producing near-maximal NKA inhibition, providing initial evidence of a favorable cardiac safety profile. Together, these findings identify Ro 41-0960 as an NKA inhibitor with a distinct chemical scaffold and provide a framework for the development of non-steroidal NKA modulators.