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

Biochem Pharmacol. 2020 Jan:171:113679. doi: 10.1016/j.bcp.2019.113679. Epub 2019 Oct 24.

Abstract

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.

Keywords: Bufadienolides; Cardenolides; Conformations; Kinetics; Na/K-ATPase; Structure-activity relationship.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bufanolides / chemistry*
  • Bufanolides / metabolism
  • Bufanolides / pharmacology
  • Cardenolides / chemistry*
  • Cardenolides / metabolism
  • Cardenolides / pharmacology
  • Cardiotonic Agents / chemistry
  • Cardiotonic Agents / metabolism
  • Cardiotonic Agents / pharmacology
  • Digoxin / chemistry
  • Digoxin / metabolism
  • Digoxin / pharmacology
  • Kidney / enzymology*
  • Kidney / metabolism
  • Kinetics
  • Molecular Structure
  • Ouabain / chemistry
  • Ouabain / metabolism
  • Ouabain / pharmacology
  • Protein Binding
  • Protein Conformation*
  • Sodium-Potassium-Exchanging ATPase / antagonists & inhibitors
  • Sodium-Potassium-Exchanging ATPase / chemistry*
  • Sodium-Potassium-Exchanging ATPase / metabolism
  • Structure-Activity Relationship*
  • Swine

Substances

  • Bufanolides
  • Cardenolides
  • Cardiotonic Agents
  • Ouabain
  • Digoxin
  • Sodium-Potassium-Exchanging ATPase
  • bufalin