Calotropin and corotoxigenin 3-O-glucopyranoside from the desert milkweed Asclepias subulata inhibit the Na+/K+-ATPase activity

Abstract

Na⁺/K⁺-ATPase is an essential transmembrane enzyme found in all mammalian cells with critical functions for cell ion homeostasis. The inhibition of this enzyme by several cardiotonic steroids (CTS) has been associated with the cytotoxic effect on cancer cell lines of phytochemicals such as ouabain and digitoxin. This study evaluated the inhibitory capacity of cardenolides calotropin and corotoxigenin 3-O-glucopyranoside (C3OG) from Asclepias subulata over the Na⁺/K⁺-ATPase activity in vitro and silico. The inhibitory assays showed that calotropin and C3OG decreased the Na⁺/K⁺-ATPase activity with IC₅₀ values of 0.27 and 0.87 μM, respectively. Furthermore, the molecules presented an uncompetitive inhibition on Na⁺/K⁺-ATPase activity, with K_i values of 0.2 μM to calotropin and 0.5 μM to C3OG. Furthermore, the molecular modeling indicated that calotropin and C3OG might interact with the Thr⁷⁹⁷ and Gln¹¹¹ residues, considered essential to the interaction with the Na⁺/K⁺-ATPase. Besides, these cardenolides can interact with amino acid residues such as Phe⁷⁸³, Leu¹²⁵, and Ala³²³, to establish hydrophobic interactions on the binding site. Considering the results, these provide novel evidence about the mechanism of action of cardenolides from A. subulata, proposing that C3OG is a novel cardenolide that deserves further consideration for in vitro cellular antiproliferative assays and in vivo studies as an anticancer molecule.

Keywords: ATPase activity; Asclepias subulata; Cardenolides; Uncompetitive inhibition.

Figure 1. Structural nomenclature of cardiac glycosides.

(A) Cardenolides. (B) Bufadienolides.

Figure 2. Structure of the cardenolides used for molecular modeling.

(A) Calotropin. (B) Corotoxigenin 3-O-glucopyranoside.

Figure 3. Inhibitory effect of cardenolides on brain porcine Na⁺/K⁺-ATPase enzymatic activity.

Non-linear regression analysis of a concentration-response curve model estimates the inhibitory concentration at 50% (IC₅₀). Green triangles depict results of calotropin and orange squares depict resullts of C3OG. The data come from three independent experiments performed in triplicate. The solid lines represent the fitted model.

Figure 4. Michaelis-Menten kinetics for the substrate (ATP) on porcine brain Na⁺/K⁺-ATPase.

The data were fitted to a Michaelis-Menten kinetics model using a non-linear regression model (R² = 0.97). All substrate concentrations were evaluated by triplicate.

Figure 5. Mode of inhibition of cardenolides over Na⁺/K⁺-ATPase.

(A) Michaelis-Menten modeling of calotropin inhibition at 0.1 μM (green squares), 1.0 μM (green triangles), and without inhibitor (blue circles). (B) Lineweaver-Burk plot of the data shown in panel A. (C) Michaelis-Menten modeling of C3OG inhibition at 0.1 μM (orange squares), 1.0 μM (orange triangles), and without inhibitor (blue circles). (D) Lineweaver-Burk plot of the data shown in panel C. Data were determined by three independent assays with three replicates.

Figure 6. Molecular docking of reference ligand used in molecular modeling with the receptor Na⁺/K⁺-ATPase (PDB 4HYT).

(A) Structure of ouabain. (B) 2D interaction map of reference ligand ouabain (white) with the receptor Na⁺/K⁺-ATPase. (C) Molecular modeling of reference ligand ouabain with the receptor Na⁺/K⁺-ATPase. A green dash line marked hydrogen bonds. A pink dash line marked hydrophobic interactions.

Figure 7. Molecular docking of Na⁺/K⁺-ATPase and the ligand calotropin (blue).

(A) Molecular modeling of cardenolide calotropin and the Na⁺/K⁺-ATPase. (B) 2D interaction map of cardenolide calotropin as a ligand with the Na⁺/K⁺-ATPase as the receptor. A green dash line marked hydrogen bonds. A pink dash line marked hydrophobic interactions.

Figure 8. Molecular docking of Na⁺/K⁺-ATPase and the ligand C3OG (orange).

(A) Molecular modeling of C3OG and the Na⁺/K⁺-ATPase. (B) 2D interaction map of C3OG as a ligand into the Na⁺/K⁺-ATPase as the receptor. Hydrogen bonds were represented as a green dash line. Hydrophobic interactions were marked by the pink and purple dash lines.