Cisplatin is the most widely used chemotherapeutic drug for the treatment of various types of cancer; however, its administration brings also numerous side effects. It was demonstrated that cisplatin can inhibit the Na+/K+-ATPase (NKA), which can explain a large part of the adverse effects. In this study, we have identified five cysteinyl residues (C452, C456, C457, C577, and C656) as the cisplatin binding sites on the cytoplasmic loop connecting transmembrane helices 4 and 5 (C45), using site-directed mutagenesis and mass spectrometry experiments. The identified residues are known to be susceptible to glutathionylation indicating their involvement in a common regulatory mechanism.

• Keywords
• C45 loop, Na+/K+-ATPase, binding site, cisplatin, cysteine mutants, sodium pump,
• MeSH
• Cisplatin chemistry   pharmacology   MeSH
• Cysteine antagonists & inhibitors   metabolism   MeSH
• Cytoplasm drug effects   metabolism   MeSH
• Mass Spectrometry MeSH
• Mutagenesis, Site-Directed MeSH
• Mice MeSH
• Antineoplastic Agents chemistry   pharmacology   MeSH
• Molecular Dynamics Simulation MeSH
• Sodium-Potassium-Exchanging ATPase antagonists & inhibitors   genetics   metabolism   MeSH
• Binding Sites drug effects   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cisplatin MeSH
• Cysteine MeSH
• Antineoplastic Agents MeSH
• Sodium-Potassium-Exchanging ATPase MeSH

We examined the inhibitory effects of three flavonolignans and their dehydro- derivatives, taxifolin and quercetin on the activity of the Na(+)/K(+)-ATPase (NKA). The flavonolignans silychristin, dehydrosilychristin and dehydrosilydianin inhibited NKA with IC50 of 110 ± 40 μM, 38 ± 8 μM, and 36 ± 14 μM, respectively. Using the methods of molecular modeling, we identified several possible binding sites for these species on NKA and proposed the possible mechanisms of inhibition. The binding to the extracellular- or cytoplasmic C-terminal sites can block the transport of cations through the plasma membrane, while the binding on the interface of cytoplasmic domains can inhibit the enzyme allosterically. Fluorescence spectroscopy experiments confirmed the interaction of these three species with the large cytoplasmic segment connecting transmembrane helices 4 and 5 (C45). The flavonolignans are distinct from the cardiac glycosides that are currently used in NKA treatment. Because their binding sites are different, the mechanism of inhibition is different as well as the range of active concentrations, one can expect that these new NKA inhibitors would exhibit also a different biomedical actions than cardiac glycosides.

• Keywords
• Na+/K+-ATPase, binding sites, flavonolignans, inhibition, sodium pump,
• Publication type
• Journal Article MeSH