RESUMEN
Sirtuin2 (Sirt2) with its NAD+-dependent deacetylase and defatty-acylase activities plays a central role in the regulation of specific cellular functions. Dysregulation of Sirt2 activity has been associated with the pathogenesis of many diseases, thus making Sirt2 a promising target for pharmaceutical intervention. Herein, we present new high affinity Sirt2 selective Sirtuin-Rearranging Ligands (SirReals) that inhibit both Sirt2-dependent deacetylation and defatty-acylation in vitro and in cells. We show that simultaneous inhibition of both Sirt2 activities results in strongly reduced levels of the oncoprotein c-Myc and an inhibition of cancer cell migration. Furthermore, we describe the development of a NanoBRET-based assay for Sirt2, thereby providing a method to study cellular target engagement for Sirt2 in a straightforward and accurately quantifiable manner. Applying this assay, we could confirm cellular Sirt2 binding of our new Sirt2 inhibitors and correlate their anticancer effects with their cellular target engagement.
RESUMEN
After traumatic injury to the central nervous system (CNS), various cytokines orchestrate the physiological responses of injured neurons and glial cells. The control of these intercellular signals is of major interest from a medical point of view. Since the transcriptional activator retinoic acid (RA) is known to regulate gene expression of cytokines in various cell culture systems we investigated the role of RA signaling in glial cells. The transcriptional activity of RA-induced genes is largely determined by the distribution of RA, which in turn depends on the local oxidation of retinaldehyde (RAL). This is synthesized from retinol or internalized as a component of vitamin A. Using high-pressure liquid chromatography and an RA-sensitive reporter cell line, we showed that OLN-93 cells, which serve as a model system for CNS oligodendrocytes, convert all-trans-RAL to the biologically active form all-trans-RA, but neither oxidize 9-cis-RAL nor isomerize RA enzymatically. The oligodendrocyte cell line expresses a cytosolic aldehyde dehydrogenase with an apparent molecular weight of 54-57 kDa and pI of 5.3-5.7. As indicated by a zymography bioassay, this enzyme is responsible for RA synthesis. The reaction requires NAD+ as cosubstrate and can be inhibited by disulfiram and citral. No other RA-producing enzyme activities were detected. These findings are in accordance with a putative role for retinoid signaling in neuroglial interactions in the CNS.