Cysteine Modification by Ebselen Reduces the Stability and Cellular Levels of 14-3-3 Proteins

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Authors

WALOEN Kai JUNG-KC Kunwar VECCHIA Elisa D. PANDEY Sunil GAŠPARIK Norbert DOSKELAND Anne PATIL Sudarshan KLEPPE Rune HRITZ Jozef NORTON William H J MARTINEZ Aurora HAAVIK Jan

Year of publication 2021
Type Article in Periodical
Magazine / Source Molecular Pharmacology
MU Faculty or unit

Central European Institute of Technology

Citation
Web https://doi.org/10.1124/molpharm.120.000184
Doi http://dx.doi.org/10.1124/molpharm.120.000184
Keywords TYROSINE-HYDROXYLASE; STRUCTURAL BASIS; PHOSPHORYLATION; 14-3-3-PROTEINS; IDENTIFICATION; PROLIFERATION; THIMEROSAL; INHIBITOR; OXIDATION; TARGETS
Description 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 14-3-3?. Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ?, ?, ?, and ?. Ebselen bonding decreased 14-3-3? binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3? (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.
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