Conformational states of the pig kidney Na+/K+-ATPase differently affect bufadienolides and cardenolides: A directed structure-activity and structure-kinetics study.

There is a renewed interest in the Na+/K+-ATPase (NKA, EC 3.6.3.9) either as a target for new therapeutic uses or for understanding the putative pathophysiological role of its mammalian endogenous ligands. Recent data indicate that bufalin binds to the pig kidney NKA in a way different from ouabain and digoxin, raising the question of a putative class difference between bufadienolides and cardenolides. The purpose of this work was to perform a study of the relationship between structure and both activity and kinetics, focusing mainly on the influence of the lactone ring in C17 (5 vs. 6 membered), the effect of C14-15 cyclization and the carbohydrate moiety in C3. We compared the potency of fourteen related cardiotonic steroids (CTS) for inhibition of the cycling pig kidney NKA in two different concentrations of K+, as well as the affinity for binding to the E2P conformation of the enzyme (Mg-Pi medium) and the potency for inhibiting the E2[2K] conformation of the NKA (K+-pNPPase activity). Cardenolides were clearly sensitive to the antagonistic effect of high K+ concentrations whereas bufadienolides were not or less sensitive. The C14-15 cyclization observed in some bufadienolides, such as resibufogenin and marinobufagin, caused a drastic fall in the affinity for binding to the NKA in the E2P conformation and increased the velocity of K+-pNPPase inhibition. The absence of a carbohydrate moiety in C3 increased the velocity of inhibition. Cardenolides were much more dependent on the E2P conformation for binding than bufadienolides since their ratios of E2[2K] IC50 to E2P Ki were higher than for bufadienolides. Therefore, the present data established the remarkable influence of C14-15 cyclization and of the carbohydrate moiety in C3 on both affinity and kinetics of CTS and indicate that, as a class, bufadienolides would harbor qualitative differences from cardenolides with respect to the NKA conformations to which they can bind.

Revisiting the binding kinetics and inhibitory potency of cardiac glycosides on Na+,K+-ATPase (α1ß1): Methodological considerations.

INTRODUCTION: Ouabain and digoxin are classical inhibitors of the Na+,K+-ATPase. In addition to their conventional uses as therapeutic agents or experimental tools there is renewed interest due to evidence suggesting they could be endogenous hormones. Somewhat surprisingly, different publications show large discrepancies in potency for inhibiting Na+,K+-ATPase activity (IC50), particularly for the slow binding inhibitors, ouabain and digoxin. METHODS: Using purified pig kidney Na+,K+-ATPase (α1ß1FXYD2) and purified detergent-soluble recombinant human Na+,K+-ATPase (α1ß1FXYD1) we have re-evaluated binding and inhibition kinetics and effects of K+ concentration for ouabain, digoxin, ouabagenin and digoxigenin. RESULTS: We demonstrate unequivocally that for slow binding inhibitors, ouabain and digoxin, long incubation times (≥60 min at 37 °C) are required to avoid under-estimation of potency and correctly determine inhibition (IC50 around 100-200 nM at 5 mM K+) contrary to what occurs when pre-incubation of the drugs without ATP is followed by a short incubation time. By contrast, for the rapidly bound inhibitors, ouabagenin and digoxigenin, short incubation times suffice (<10 min). The strong reduction of inhibitory potency observed at high un-physiological K+ concentrations (≥5 mM) also explained the low potency reported by some authors. DISCUSSION: The data resolve discrepancies in the literature attributable to sub-optimal assay conditions. Similar IC50 values are obtained for pig kidney and recombinant human Na+,K+-ATPase, showing that inhibitory potencies are not determined by the species difference (pig versus human) or environment (membrane-bound versus detergent-soluble) of the Na+,K+-ATPase. The present methodological considerations are especially relevant for drug development of slow binding inhibitors.

Validation of a Na+-shift binding assay for estimation of the intrinsic efficacy of ligands at the A2A adenosine receptor.

INTRODUCTION: Determination of the intrinsic efficacy of ligands at the A2A receptor is important for selecting drug candidates, e.g. in the case of inflammatory diseases where agonists are searched for or in Parkinson disease (antagonists). METHODS: Three functional binding assays were compared with up to seven ligands with different efficacies: the GTP-shift method based on the decrease of affinity observed with agonists when GTP is added to the competition binding assay; the Ki ratio method based on the different affinity states of the receptor when using an agonist or antagonist radioligand and the Na+-shift assay based on the difference of affinity of agonists when tested in a medium containing a divalent cation (50mM MgCl2) favoring the G protein coupled agonist-receptor complex or sodium (100mM NaCl) as negative allosteric modulator. RESULTS: The Na+-shift assay proposed herein successfully discriminated the full agonists CGS21680, NECA and adenosine (IC50 ratio=13-14) from the weak inverse agonists ZM241385 and IBMX (IC50 ratio=0.85) and the partial agonists LUF5834 and regadenoson (IC50 ratios equal to 3 and 10, respectively). DISCUSSION: We conclude that the Na+-shift assay proposed herein for the A2A receptors has been validated and represents a rapid, economic and efficient functional binding assay to be used in a drug development program for early estimation of the intrinsic efficacy of hits.