Impact of Time, Temperature, and Conformational Changes on the Characterization of Cardiotonic Steroid-Na⁺/K⁺-ATPase...

Cardiotonic steroids (CTS) have gained prominence due to various mechanisms of action proposed or demonstrated to explain their effects, including their known classical and non-classical signaling pathways mediated by Na⁺/K⁺-ATPase (NKA) binding. For the characterization, interaction kinetics have been used to better translate in vivo efficacy and selectivity. In addition, temperature effects assessed by thermodynamic analysis provide deeper insight into intrinsic efficacy. However, the impact of conformational changes on both kinetic and thermodynamic parameters of CTS binding has been largely overlooked. This review discusses how these kinetic effects relate to efficacy and selectivity in the experimental characterization of CTS-NKA interactions. We focus on the most straightforward and high-throughput methods for describing CTS-NKA interaction, such as enzymatic activity inhibition, looking at dephosphorylation activity of NKA cycle, and compare them with gold-standard methods and its limitations. Finally, we include some statistical considerations for data analysis and future perspectives. This will provide better insight into the efficacy of this class of inhibitors/agonists targeting the NKA in drug discovery.