Three-Dimensional Quantitative Structure−Activity Relationship Study of the Inhibition of Na⁺,K⁺-ATPase by Cardiotonic Steroids Using Comparative Molecular Field Analysis

Abstract

Na(+),K(+)-ATPase is a transmembrane protein that transports sodium and potassium ions across cell membranes during an activity cycle that uses the energy released by ATP hydrolysis. Cardiotonic steroids (digitalis) inhibit this activity and consequently produce a positive inotropic response in the heart. To identify the structural features of the steroids that are important for this inhibition, we have tested the inhibitory properties of 47 cardiotonic and hormonal steroids and developed a three-dimensional quantitative structure-activity relationship (3D-QSAR) model for the inhibition of Na(+),K(+)-ATPase using comparative molecular field analysis (CoMFA). We also developed a 3D-QSAR model for the binding of digoxin to the murine anti-digoxin monoclonal antibody (mAb) 26-10 because we have previously shown that the environment of the binding sites of 26-10 and the enzyme are similar (Kasturi et al. (1998) Biochemistry 37, 6658-6666). These statistically predictive 3D-QSAR models indicate that both binding sites are about 20 A long and have a close fit or complementarity about the beta side of the lactone ring of digitalis. Furthermore, steric bulk about the lactone ring and the alpha sugar may be critical for drug binding. However, the binding site of Na(+),K(+)-ATPase differs from that of mAb in that it has a greater number of electrostatic interactions along the alpha-sugar, steroid, and lactone moieties. In addition, the availability of the structure of the 26-10 Fab-digoxin complex (Jeffrey et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10310-10314) enabled us to compare the CoMFA-derived contour maps with the known locations for amino acid residues comprising the mAb ligand binding site.

Characterization of the Neutralizing Activity of Digoxin-Specific Fab toward Ouabain-Like Steroids

“…Using 28 steroid Na ϩ /K ϩ ATPase inhibitors, it was found that adding steric bulk and/or electronegativity around the sugar attached to position 3 increased inhibitory activity. The importance of hydroxylation of position 5 in affecting the spatial orientation of gycosides in position 3 has recently been show also using molecular field analysis (Farr et al, 2002b). A greater range of inhibitory activity was seen in this study compared with Farr et al (2002b).…”

The cardiotonic steroid hormone marinobufagenin induces renal fibrosis: implication of epithelial-to-mesenchymal transition

“…*P Ͻ 0.05, **P Ͻ 0.001 vs. control (n ϭ 9 from 3 independent experiments). tracellular signal through the same molecule, the ␣-subunit of Na ϩ -K ϩ -ATPase (16,21). MBG and ouabain belong to two different subclasses of CTSs: bufadienolides, containing a five-membered lactone ring, and cardienolides with six-membered lactone, respectively.…”

“…MBG and ouabain belong to two different subclasses of CTSs: bufadienolides, containing a five-membered lactone ring, and cardienolides with six-membered lactone, respectively. The structure of lactone ring moiety of CTS has been shown to determine binding and inhibitory activities of CTSs (21).…”

“…Moreover, elevated levels of endogenous CTSs including marinobufagenin (MBG) have been associated with various pathological conditions: essential hypertension (4,24,73), preeclampsia (3,45,83), experimental diabetes (7), uremic cardiomyopathy (33), and cardiac failure (20,48,49,66,74). MBG, like other CTSs, binds to the extracellular domain of the ␣-subunit of Na ϩ -K ϩ -ATPase (16,21), which, in addition to its well-known function of maintaining cellular electrochemical balance through pumping sodium and potassium ions, can act as a typical membrane receptor (14,28,35,55,56,73,74,84,85,88).…”

New Molecular Determinants Controlling the Accessibility of Ouabain to Its Binding Site in Human Na,K-ATPase α Isoforms

“…So far, only ␣1 isoforms have been studied with respect to the localization of the binding site and the molecular determinants of the association and dissociation kinetics of cardiac glycosides. Experimental and modeling data suggest that cardiac glycosides bind to the extracellular surface of the ␣-subunit (Croyle et al, 1997;Middleton et al, 2000;Farr et al, 2002) and that the physical binding site for cardiac glycosides may be formed by the M3/M4 and M5/M6 hairpins (Koenderink et al, 2000). The association rate of ouabain seems to be dependent on the steroid moiety, whereas the dissociation rate depends both on the steroid and the sugar moieties (Yoda, 1974;Kawamura et al, 2001).…”