Abstract
The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.
Highlights
The 14-3-3 proteins constitute a family of highly conserved acidic proteins that form homo- and heterodimers between isoforms
The canonical binding site for protein-protein interaction (PPI) in 14-3-3 is situated on the concave surface of each subunit, and binding of a partner protein to 14-3-3 proteins is typically dependent on a phosphorylated Ser or Thr flanked by Arg and Pro residues (Aitken, 2006; Obsil and Obsilova, 2011)
To identify compounds capable of binding to 14-3-3f, a differential scanning fluorimetry (DSF)-based high-throughput screen was carried out using an established protocol for identification of binders to protein targets (Niesen et al, 2007; Aubi et al, 2015; Urbaneja et al, 2017)
Summary
The 14-3-3 proteins constitute a family of highly conserved acidic proteins that form homo- and heterodimers between isoforms. 14-3-3 proteins have no enzymatic activity, their binding to phosphorylated partners exerts chaperone as well as regulatory functions (van Heusden, 2005). 14-3-3 proteins are involved in many vital cellular functions, such as transcription, intracellular trafficking, cytoskeletal structure, metabolic regulation, and apoptosis (Aitken, 2006; Obsil and Obsilova, 2011). The canonical binding site for protein-protein interaction (PPI) in 14-3-3 is situated on the concave surface of each subunit, and binding of a partner protein to 14-3-3 proteins is typically dependent on a phosphorylated Ser or Thr flanked by Arg and Pro residues (Aitken, 2006; Obsil and Obsilova, 2011)
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