Selectivity analyses of γ-benzylidene digoxin derivatives to different Na,K-ATPase α isoforms: a molecular docking approach

<p>Digoxin and other cardiotonic steroids (CTS) exert their effect by inhibiting Na,K-ATPase (NKA) activity. CTS bind to the various NKA isoforms that are expressed in different cell types, which gives CTS their narrow therapeutic index. We have synthesised a series of digoxin derivatives (γ-Benzylidene digoxin derivatives) with substitutions in the lactone ring (including non-oxygen and ether groups), to obtain CTS with better NKA isoform specificity. Some of these derivatives show some NKA isoform selective effects, with BD-3, BD-8, and BD-13 increasing NKA α2 activity, BD-5 inhibiting NKA α1 and NKA α3, BD-10 reducing NKA α1, but stimulating NKA α2 and α3; and BD-14, BD-15, and BD-16 enhancing NKA α3 activity. A molecular-docking approach favoured NKA isoform specific interactions for the compounds that supported their observed activity. These results show that BD compounds are a new type of CTS with the capacity to target NKA activity in an isoform-specific manner.</p

Influence of Fe³⁺ on the activity of Na,K-ATPase.

<p>(A) Activity of Na,K-ATPase in erythrocyte membranes of individuals with or without iron overload: n = 24 (case) and n = 12 (control), p<0.0001 (Mann Whitney). (B) Activity of Na,K-ATPase in erythrocyte membranes of subjects with or without iron overload, stratified by age group: n = 12 (<50 years), n = 06 (control <50 years), n = 12 (> = 50 years), n = 06 (Control > = 50 years). p = 0.002 (case x control <50 years), p = 0.005 (case x control > = 50 years), Student's t test. All experiments were performed in triplicates and the line in bars represented the mean and the error bar denotes SEM.</p

Main clinical manifestations reported by patients with hyperferritinemia grouped by age.

<p>* X²</p><p>^<b>†</b> n = 10</p><p>^<b>‡</b> n = 11</p><p>Main clinical manifestations reported by patients with hyperferritinemia grouped by age.</p

Influence of Fe³⁺ on the activity of Na,K-ATPase.

<p>(A) Activity of Na,K-ATPase in erythrocyte membranes of individuals with or without iron overload: n = 24 (case) and n = 12 (control), p<0.0001 (Mann Whitney). (B) Activity of Na,K-ATPase in erythrocyte membranes of subjects with or without iron overload, stratified by age group: n = 12 (<50 years), n = 06 (control <50 years), n = 12 (> = 50 years), n = 06 (Control > = 50 years). p = 0.002 (case x control <50 years), p = 0.005 (case x control > = 50 years), Student's t test. All experiments were performed in triplicates and the line in bars represented the mean and the error bar denotes SEM.</p

Iron overload effect on oxidative stress.

<p>(A) Content of Fe³⁺ in the residual plasma of individuals with or without iron overload: n = 24 (case) and n = 14 (control), p = 0.087 (Mann Whitney). (B) Lipid peroxidation in subjects with or without iron overload: n = 14 (case) and n = 14 (control), p<0.0001 (Student's t test). All experiments were performed in triplicate and the line in bars represented the mean and the error bar denotes SEM.</p

Influence of iron overload on lipid profile of the erythrocyte plasma membrane.

<p>(A) Total phospholipids: n = 23 (case) and n = 14 (control), p = 0.013 (Student's t test). (B) Total phospholipids of patients with iron overload, stratified by age: n = 11 (<50 years) and n = 12 (> = 50 years), p = 0.554 (Student's t test). (C) Total membrane cholesterol: n = 23 (case) and n = 14 (control), p<0.001 (Student's t test). (D) Total membrane cholesterol of patients with iron overload, stratified by age: n = 11 (<50 years) and n = 12 (> = 50 years), p = 0.241 (Student's t test). All experiments were performed in triplicate and the line in bars represented the mean and the error bar denotes SEM.</p

H₂O₂ and iron effect on red blood cell.

<p>Red blood cell from health donators were incubated with 0.8 mM H₂O₂ or 1 μM FeCl₃ for 24h at 4°C. This sample was used to obtained: (A) plasma TBARS levels, (B) the Na,K-ATPase activity of membrane preparation and the total levels of (C) cholesterol and (D) phospholipids. All experiments were performed in triplicates and the line in bars represented the mean and the error bar denotes SEM, *p <0.05; results are significantly different from controls, as evaluated by ANOVA test.</p

21-Benzylidene Digoxin: A Proapoptotic Cardenolide of Cancer Cells That Up-Regulates Na,K-ATPase and Epithelial Tight Junctions

<div><p>Cardiotonic steroids are used to treat heart failure and arrhythmia and have promising anticancer effects. The prototypic cardiotonic steroid ouabain may also be a hormone that modulates epithelial cell adhesion. Cardiotonic steroids consist of a steroid nucleus and a lactone ring, and their biological effects depend on the binding to their receptor, Na,K-ATPase, through which, they inhibit Na⁺ and K⁺ ion transport and activate of several intracellular signaling pathways. In this study, we added a styrene group to the lactone ring of the cardiotonic steroid digoxin, to obtain 21-benzylidene digoxin (21-BD), and investigated the effects of this synthetic cardiotonic steroid in different cell models. Molecular modeling indicates that 21-BD binds to its target Na,K-ATPase with low affinity, adopting a different pharmacophoric conformation when bound to its receptor than digoxin. Accordingly, 21-DB, at relatively high µM amounts inhibits the activity of Na,K-ATPase α₁, but not α₂ and α₃ isoforms. In addition, 21-BD targets other proteins outside the Na,K-ATPase, inhibiting the multidrug exporter Pdr5p. When used on whole cells at low µM concentrations, 21-BD produces several effects, including: 1) up-regulation of Na,K-ATPase expression and activity in HeLa and RKO cancer cells, which is not found for digoxin, 2) cell specific changes in cell viability, reducing it in HeLa and RKO cancer cells, but increasing it in normal epithelial MDCK cells, which is different from the response to digoxin, and 3) changes in cell-cell interaction, altering the molecular composition of tight junctions and elevating transepithelial electrical resistance of MDCK monolayers, an effect previously found for ouabain. These results indicate that modification of the lactone ring of digoxin provides new properties to the compound, and shows that the structural change introduced could be used for the design of cardiotonic steroid with novel functions.</p></div

21-BD regulates tight junctions.

<p>MDCK cells were cultured in transwell permeable supports and treated with 5, 10 and 50 µM 21-BD. (A) TER was measured as a function of time. The control TER data (white circles, dotted line) averaged 183±8 Ω.cm² (n = 13) and were normalized to 100%. 5 and 10 µM 21-BD provoke transient small increases of TER, while 50 µM 21-BD causes a stronger and a sustained TER increase (red circles). (B) MDCK cells were incubated 48 h with different concentrations of 21-BD (red symbols) or digoxin (green symbols). mRNA cell content of claudins -4 (circles) and -2 (triangles) were measured by quantitative real time PCR. (C) Protein cell content of the tight junction integral membrane proteins claudins -4 and -2 and the membrane-associated protein ZO-1 as a function of 21-BD concentration in the media for 48 h. Images from the left part of the figure C are representative immunoblots and the graph in the right part is the statistical analysis.</p

21-BD induces apoptosis in HeLa and CHO-K1 cells.

<p>(A) Score value obtained from the comet assay of CHO-K1 cells incubated 24 h with 21-BD at different concentrations (red circles). (B) Micronucleated cells percentage of CHO-K1 cultures incubated with 21-BD at different concentrations for 24 h. A 24 h incubation with 0.4 mM Methyl methanesulfonate (MMS) was used as a control (A, B, blue circles). (C) Apoptotic and necrotic HeLa cells after 24 h of incubation in control media (white bars), media with 50 µM 21-BD (red bars) or 2 µM digoxin (green bars) for 24 h. Apoptosis and necrosis were detected by flow cytometry ussing an annexin-V translocation assay and the incorporation of propidium iodide in to the nucleus, respectively. <i>P</i><0.01.</p