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 IC 50 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 (IC 50 > 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.