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

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.

Druggable genome in attention deficit/hyperactivity disorder and its co-morbid conditions. New avenues for treatment.

Attention-Deficit/Hyperactivity Disorder (ADHD) is a common neurodevelopmental disorder with only symptomatic care available. Genome-wide association (GWA) studies can provide a starting point in the search for novel drug targets and possibilities of drug repurposing. Here, we explored the druggable genome in ADHD by utilising GWA studies on ADHD and its co-morbid conditions. First, we explored whether the genes targeted by current ADHD drugs show association with the disorder and/or its co-morbidities. Second, we aimed to identify genes and pathways involved in the biological processes underlying ADHD that can be targeted by pharmacological agents. These ADHD-associated druggable genes and pathways were also examined in co-morbidities of ADHD, as commonalities in their aetiology and management may lead to novel pharmacological insights. Strikingly, none of the genes encoding targets of first-line pharmacotherapeutics for ADHD were significantly associated with the disorder, suggesting that FDA-approved ADHD drugs may act through different mechanisms than those underlying ADHD. In the examined druggable genome, three loci on chromosomes 1, 4 and 12 revealed significant association with ADHD and contained nine druggable genes, five of which encode established drug targets for malignancies, autoimmune and neurodevelopmental disorders. To conclude, we present a framework to assess the druggable genome in a disorder, exemplified by ADHD. We highlight signal transduction and cell adhesion as potential novel avenues for ADHD treatment. Our findings add to knowledge on known ADHD drugs and present the exploration of druggable genome associated with ADHD, which may offer interventions at the aetiological level of the disorder.

Tyrosine and tryptophan hydroxylases as therapeutic targets in human disease.

INTRODUCTION: The ancient and ubiquitous monoamine signalling molecules serotonin, dopamine, norepinephrine, and epinephrine are involved in multiple physiological functions. The aromatic amino acid hydroxylases tyrosine hydroxylase (TH), tryptophan hydroxylase 1 (TPH1), and tryptophan hydroxylase 2 (TPH2) catalyse the rate-limiting steps in the biosynthesis of these monoamines. Genetic variants of TH, TPH1, and TPH2 genes are associated with neuropsychiatric disorders. The interest in these enzymes as therapeutic targets is increasing as new roles of these monoamines have been discovered, not only in brain function and disease, but also in development, cardiovascular function, energy and bone homeostasis, gastrointestinal motility, hemostasis, and liver function. Areas covered: Physiological roles of TH, TPH1, and TPH2. Enzyme structures, catalytic and regulatory mechanisms, animal models, and associated diseases. Interactions with inhibitors, pharmacological chaperones, and regulatory proteins relevant for drug development. Expert opinion: Established inhibitors of these enzymes mainly target their amino acid substrate binding site, while tetrahydrobiopterin analogues, iron chelators, and allosteric ligands are less studied. New insights into monoamine biology and 3D-structural information and new computational/experimental tools have triggered the development of a new generation of more selective inhibitors and pharmacological chaperones. The enzyme complexes with their regulatory 14-3-3 proteins are also emerging as therapeutic targets.

Lectin-like bacteriocins from Pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor.

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of D-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing D-rhamnose and not D-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.