The antitumor effect of cardiotonic steroids (CTS) has stimulated the search for new methods to evaluate both kinetic and thermodynamic aspects of their binding to Na⁺/K⁺-ATPase (IUBMB Enzyme Nomenclature). We propose a real-time assay based on a chromogenic substrate for phosphatase activity (pNPPase activity), using only two concentrations with an inhibitory progression curve, to obtain the association rate (k_on), dissociation rate (k_off), and equilibrium (K_i) constants of CTS for the structure-kinetics relationship in drug screening. We show that changing conditions (from ATPase to pNPPase activity) resulted in an increase of K_i of the cardenolides digitoxigenin, essentially due to a reduction of k_on. In contrast, the K_i of the structurally related bufadienolide bufalin increased much less due to the reduction of its k_off partially compensating the decrease of its k_on. When evaluating the kinetics of 15 natural and semisynthetic CTS, we observed that both k_on and k_off correlated with K_i (Spearman test), suggesting that differences in potency depend on variations of both k_on and k_off. A rhamnose in C3 of the steroidal nucleus enhanced the inhibitory potency by a reduction of k_off rather than an increase of k_on. Raising the temperature did not alter the k_off of digitoxin, generating a ΔH^‡ (k_off) of −10.4 ± 4.3 kJ/mol, suggesting a complex dissociation mechanism. Based on a simple and inexpensive methodology, we determined the values of k_on, k_off, and K_i of the CTS and provided original kinetics and thermodynamics differences between CTS that could help the design of new compounds. SIGNIFICANCE STATEMENT This study describes a fast, simple, and cost-effective method for the measurement of phosphatase pNPPase activity enabling structure-kinetics relationships of Na⁺/K⁺-ATPase inhibitors, which are important compounds due to their antitumor effect and endogenous role. Using 15 compounds, some of them original, this study was able to delineate the kinetics and/or thermodynamics differences due to the type of sugar and lactone ring present in the steroid structure.

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. Graphical Abstract Drug-Target Interaction with Deep and Complex Binding Site.