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 (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.