Abstract
The 14-3-3 proteins constitute a family of regulatory adapter proteins with many binding partnersand biological functions, and 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. C25 was identified as the preferential site of ebselen interaction in vitro, whereas modification of C94 was mainly responsible for the protein destabilization. At therapeutic relevant concentrations ebselen caused a decrease of 14-3-3 levels in both SHSY5Y cells and zebrafish brain, accompanied with an increased degradation by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed a freezing phenotype and reduced mobility, resembling the effects of lithium, in line with the proposed action of ebselen as a potentially safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, including cancer, neuropsychiatric disorders and infectious diseases, including Covid-19. Its pleiotropic actions are attributed to antioxidant effects, and the 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 virus infection Covid-19, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering 14-3-3 destabilization and degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral and anti-neoplastic effects of this drug.
- brain/CNS
- covalent drug binding
- mood stabilizers
- neuroblastoma
- neurodegeneration
- neurotransmitters
- Oxidative stress/antioxidants
- Protein targets
- © 2020 The Authors. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited and is not used for commercial purposes.