Simplified Method for Kinetic and Thermodynamic Screening of Cardiotonic Steroids Through the K + -Dependent Phosphatase Activity of Na + /K + -ATPase with Chromogenic pNPP Substrate .

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 (NKA, EC 3.6.3.9). 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 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 semi-synthetic 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 Rising the temperature did not alter the k off of digitoxin, generating a ∆H ‡ (k off ) of -10.4 {plus minus} 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 We described 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, we were able to delineate the kinetics and/or thermodynamics differences due to the type of sugar and lactone ring present in the steroid structure.