Discovery of Ro 41‐0960, a Non‐Steroidal Inhibitor of the Na + /K + ‐ATPase

Figures

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  FIGURE 1. Concentration‐dependent inhibition of NKA by Ro 41‐0960. ATPase activity of purified NKA (α1 isoform) is shown as a function of Ro 41‐0960 concentration. Enzyme activity is normalized to control conditions in the absence of compound and 3 mM ATP. Data were fitted using a four‐parameter nonlinear concentration–response model with a variable slope, yielding an IC50 value of 17.9 ± 1.1 µM. The chemical structure of Ro 41‐0960 is shown. Data represent mean ± SEM from independent experiments (N = 3).

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  FIGURE 2. Effects of Ro 41‐0960 on Na+‐, K+‐, and ATP‐dependent activity of purified NKA. (A) ATPase activity as a function of NaCl concentration, measured using the coupled enzyme assay. (B) ATPase activity as a function of KCl concentration, measured using the direct phosphate‐release assay. (C) ATPase activity as a function of ATP concentration, measured using the coupled enzyme assay. In all panels, enzyme activity in the absence (control, black) and presence of Ro 41‐0960 (red) is shown. In all cases, Ro 41‐0960 was evaluated at a concentration of 18 µM. The substrate‐dependence experiments shown in each panel were performed as independent experimental series and normalized independently to the corresponding control reference activity. ATP‐dependent activities in panel C were normalized to the maximal control activity observed at 3–4 mM ATP. Data in panels A and B were fitted to a nonlinear allosteric sigmoidal model. Data in panel C were fitted to a nonlinear bell‐shaped model for the control condition and a nonlinear biphasic model in the presence of Ro 41‐0960. Data represent mean ± SEM from independent experiments (N = 3).

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  FIGURE 3. Inhibition of NKA by Ro 41‐0960 in microsomal preparations. (A) Concentration‐dependent inhibition of NKA activity measured using the coupled enzyme assay, yielding an IC50 of 10.3 ± 1.1 µM at 3 mM ATP. (B) ATP dependence of NKA activity measured using the coupled enzyme assay in the absence (control, black) and presence of 18 µM Ro 41‐0960 (red). ATP‐dependent activities were normalized to the maximal control activity observed at 3–4 mM ATP. Data in panel A were fitted using a four‐parameter nonlinear concentration–response model with a variable slope. Data in panel B were fitted to a nonlinear bell‐shaped model. Data represent mean ± SEM from independent experiments (N = 3).

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  FIGURE 4. Selective inhibition of NKA over cardiac SERCA by Ro 41‐0960 in microsomal preparations. Concentration‐dependent inhibition of NKA (red) and SERCA (black) ATPase activity measured using coupled enzyme assays under the respective assay conditions described in the experimental section. The NKA concentration‐response dataset is reproduced from Figure 3A to facilitate direct comparison with the SERCA concentration‐response data. Ro 41‐0960 inhibits NKA with an IC50 of 10.3 ± 1.1 µM at 3 mM ATP, SERCA activity is only weakly inhibited at substantially higher concentrations (IC50 > 100 µM). In both cases, data were fitted using a four‐parameter nonlinear concentration–response model with a variable slope. Data represent mean ± SEM from independent experiments (N = 3).

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  FIGURE 5. Predicted binding modes of Ro 41‐0960 in CTS‐ and nucleotide‐binding sites of NKA. Representative docking poses of Ro 41‐0960 within the CTS‐binding pocket (left; PDB: 4RES) and the nucleotide‐binding site (right; PDB: 3WGU) of pig NKA (α1 isoform). Insets show close‐up views of predicted ligand positioning and surrounding residues in each binding site. These models illustrate plausible static binding orientations used as starting configurations for subsequent MD simulations. In all panels, NKA is shown in cartoon representation, Ro 41‐0960 is shown as spheres, and interacting residues are depicted as sticks. The approximate position of the lipid bilayer is indicated by tan spheres. Extracellular and cytosolic orientations are indicated for structural reference.

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  FIGURE 6. Dynamic behavior of Ro 41‐0960 in CTS‐ and nucleotide‐binding sites during MD simulations. Ligand positional behavior across independent MD replicates in the (A) CTS‐binding pocket and (B) nucleotide‐binding site. In the CTS‐binding pocket, Ro 41‐0960 remains more positionally confined near residues contributing aromatic and hydrophobic contacts, including Phe783, Phe786, Ile780, Leu795, and Pro794. In the nucleotide‐binding site, Ro 41‐0960 samples broader poses near residues including Phe475, Arg544, Arg685, and Asn713. (C) Distribution of ligand RMSD values relative to the starting docking pose, showing lower and more narrowly distributed RMSD values in the CTS‐binding pocket than in the nucleotide‐binding site, supporting greater positional stability of Ro 41‐0960 within the CTS‐binding pocket.

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  FIGURE 7. Ro 41‐0960 does not alter impulse propagation and APD90 in human iPSC‐derived cardiomyocytes. (A) Conduction velocity and (B) action potential duration at 90% repolarization (APD90) measured in spontaneously beating human iPSC‐derived cardiomyocytes across increasing concentrations of Ro 41‐0960 (0–50 µM). Significance was evaluated using a two‐way ANOVA followed by Dunnett's post‐hoc test. No significant differences were observed relative to control conditions (ns, not significant). Data are presented as mean ± SEM with individual data points shown (N = 6).