Interactions between Cardiac Glycosides and Sodium/Potassium-ATPase:  Three-Dimensional Structure−Activity Relationship Models for Ligand Binding to the E₂-P_i Form of the Enzyme versus Activity Inhibition

Abstract

Sodium/potassium-ATPase (Na/K-ATPase) is a transmembrane enzyme that utilizes energy gained from ATP hydrolysis to transport sodium and potassium ions across cell membranes in opposite directions against their chemical and electrical gradients. Its transport activity is effectively inhibited by cardiac glycosides, which bind to the extracellular side of the enzyme and are of significant therapeutic value in the treatment of congestive heart failure. To determine the extent to which high-affinity binding of cardiac glycosides correlates with their potency in inhibiting pump activity, we determined experimentally both the binding affinities and inhibitory potencies of a series of 37 cardiac glycosides using radioligand binding and ATPase activity assays. The observed variations in key structural elements of these compounds correlating with binding and inhibition were analyzed by comparative molecular similarity index analysis (CoMSIA), which allowed a molecular level characterization and comparison of drug-Na/K-ATPase interactions that are important for ligand binding and activity inhibition. In agreement with our earlier comparative molecular field analysis studies [Farr, C. D., et al. (2002) Biochemistry 41, 1137-1148], the CoMSIA models predicted favorable inhibitor interactions primarily at the alpha-sugar and lactone ring moieties of the cardiac glycosides. Unfavorable interactions were located about the gamma-sugar group and at several positions about the steroid ring system. Whereas for most compounds a correlation between binding affinity and inhibitory potency was found, some notable exceptions were identified. Substitution of the five-membered lactone of cardenolides with the six-membered lactone of bufadienolides caused binding affinity to decline but inhibitory potency to increase. Furthermore, while the removal of ouabain's rhamnose moiety had little effect on inhibitory potency, it caused a dramatic decline in ligand binding affinity.

The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase

“…Although the results between the QN mutant and the EGPLC mutant for the various analogues differ, the IC 50 values obtained with both methods (ATPase inhibition measurements and [ 3 H]ouabain replacement studies) are, in general, rather similar for a single drug used with one of both ATPases. Our findings for Na,K-ATPase (QN) contrast with those of Paula et al [26] who carried out apparently similar studies on lamb kidney ATPase (that also contains a Gln on position 111 and an Asn on position 122) with a very large series of Amino acids that were shown to be essential for ouabain binding [13,14] are indicated in yellow. Images were created with Yasara (www.…”

“…yasara.org) Table 2 Hydrogen bonds between Na,K-ATPase (QN) and ouabain analogues Na,K-ATPase ouabain analogues. Whereas their results on Na,K-ATPase inhibition, with exception of those for strophanthidin, fit reasonably well with our findings, Paula et al [26] found for all inhibitors, except digoxin, much higher IC 50 values in ouabain replacement studies than we found in the present study (Table 3). An explanation for this discrepancy cannot yet be given.…”

“…It might be that the absence of the long hydrophilic trisdigitoxose gives this genin also the chance to penetrate more deeply into the membrane and form hydrogen bridges that could not occur when the sugar is present in the molecule. A deeper penetration into the membrane might also explain the Ouabain analogues are obtained in the present study and by Paula et al [26] relatively high affinity of Na,K-ATPase (QN) for strophanthidin and ouabagenin. The wild-type non-gastric H,K-ATPase has a very low affinity for ouabain and the five tested analogues.…”

Progesterone binding to the α1-subunit of the Na/K-ATPase on the cell surface: Insights from computational modeling

“…The amino acid sequences of the 5 external loops of the α1 isoforms of rat, sheep and X. laevis differ primarily in the 23 amino acid external loop between M1 and M2. The residues identified as determinants of cardiac glycoside binding in the sheep and human enzymes [45,46] Table 3). In the first external loop, 5 of the 23 amino acids in rat differ from sheep, 3 of which have been identified as determinants of ouabain binding.…”

Enhancing the anticancer properties of cardiac glycosides by neoglycorandomization

“…In addition to its well known cardiac activity, which is mediated by inhibition of the plasma membrane Na ϩ ͞K ϩ -ATPase (17), digitoxin has demonstrated in vitro anticancer properties (18), and patient profiling suggests the survival rate of cancer patients taking digitoxin is statistically enhanced (19,20). Cardiac glycosides were also recently noted to inhibit the expression of four genes that are overexpressed in prostate cancer cells, including transcription factors and the apoptosis inhibitor survivin (21), and to provide protective effects against polyglutamine-based diseases (22).…”