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

Abstract

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 (kon), dissociation rate (koff) and equilibrium (Ki) 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 Ki of the cardenolides digitoxigenin, essentially due to a reduction of kon In contrast, the Ki of the structurally related bufadienolide bufalin increased much less due to the reduction of its koff partially compensating the decrease of its kon When evaluating the kinetics of 15 natural and semi-synthetic CTS, we observed that both kon and koff correlated with Ki (Spearman test), suggesting that differences in potency depend on variations of both kon and koff A rhamnose in C3 of the steroidal nucleus enhanced the inhibitory potency by a reduction of koff rather than an increase of kon Rising the temperature did not alter the koff of digitoxin, generating a ∆H‡ (koff) 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 kon, koff, and Ki 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.