Sirtuins are NAD+‐dependent protein deacylases that cleave off acetyl, as well as other acyl groups, from the ε‐amino group of lysines in histones and other substrate proteins. Seven sirtuin isotypes ...(Sirt1–7) have been identified in mammalian cells. As sirtuins are involved in the regulation of various physiological processes such as cell survival, cell cycle progression, apoptosis, DNA repair, cell metabolism, and caloric restriction, a dysregulation of their enzymatic activity has been associated with the pathogenesis of neoplastic, metabolic, infectious, and neurodegenerative diseases. Thus, sirtuins are promising targets for pharmaceutical intervention. Growing interest in a modulation of sirtuin activity has prompted the discovery of several small molecules, able to inhibit or activate certain sirtuin isotypes. Herein, we give an update to our previous review on the topic in this journal (Schemies, 2010), focusing on recent developments in sirtuin biology, sirtuin modulators, and their potential as novel therapeutic agents.
Here we report the development of a proteolysis targeting chimera (PROTAC) based on the combination of the unique features of the sirtuin rearranging ligands (SirReals) as highly potent and ...isotype-selective Sirt2 inhibitors with thalidomide, a bona fide cereblon ligand. For the first time, we report the formation of a PROTAC by Cu(I)-catalyzed cycloaddition of a thalidomide-derived azide to an alkynylated inhibitor. This thalidomide-derived azide as well as the highly versatile linking strategy can be readily adapted to alkynylated ligands of other targets. In HeLa cells, our SirReal-based PROTAC induced isotype-selective Sirt2 degradation that results in the hyperacetylation of the microtubule network coupled with enhanced process elongation. Thus, our SirReal-based PROTAC is the first example of a probe that is able to chemically induce the degradation of an epigenetic eraser protein.
Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes ...the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.
Sirtuins are NAD+-dependent protein deacylases capable of cleaving off acetyl as well as other acyl groups from the ɛ-amino group of lysines in histones and other substrate proteins. They have been ...reported as promising drug targets, and thus modulators of their activity are needed as molecular tools to uncover their biological function and as potential therapeutics. Here, we present new assay formats that complement existing assays for sirtuin biochemistry and cellular target engagement. Firstly, we report the development of a homogeneous fluorescence-based activity assay using unlabelled acylated peptides. Upon deacylation, the free lysine residue reacts with fluorescamine to form a fluorophore. Secondly, using click chemistry with a TAMRA-azide on a propargylated sirtuin inhibitor, we prepared the first fluorescently labelled small-molecule inhibitor of Sirt2. This is used in a binding assay, which is based on fluorescence polarization. We used it successfully to map potential inhibitor-binding sites and also to show cellular Sirt2 engagement. By means of these new assays, we were able to identify and characterize novel Sirt2 inhibitors out of a focused library screen. The binding of the identified Sirt2 inhibitors was rationalized by molecular docking studies. These new chemical tools thus can enhance further sirtuin research.
This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology'.
Sirtuins are NAD+-dependent protein deacylases that cleave off acetyl but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Dysregulation of human ...Sirt2 (hSirt2) activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, which makes the modulation of hSirt2 activity a promising strategy for pharmaceutical intervention. The sirtuin rearranging ligands (SirReals) have recently been discovered by us as highly potent and isotype-selective hSirt2 inhibitors. Here, we present a well-defined structure–activity relationship study, which rationalizes the unique features of the SirReals and probes the limits of modifications on this scaffold regarding inhibitor potency. Moreover, we present a crystal structure of hSirt2 in complex with an optimized SirReal derivative that exhibits an improved in vitro activity. Lastly, we show cellular hyperacetylation of the hSirt2 targeted tubulin caused by our improved lead structure.
Sirtuins are NAD+‐dependent protein deacylases that cleave off acetyl groups, as well as other acyl groups, from the ɛ‐amino group of lysines in histones and other substrate proteins. Dysregulation ...of human Sirt2 activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, thus making Sirt2 a promising target for pharmaceutical intervention. Here, based on a crystal structure of Sirt2 in complex with an optimized sirtuin rearranging ligand (SirReal) that shows improved potency, water solubility, and cellular efficacy, we present the development of the first Sirt2‐selective affinity probe. A slow dissociation of the probe/enzyme complex offers new applications for SirReals, such as biophysical characterization, fragment‐based screening, and affinity pull‐down assays. This possibility makes the SirReal probe an important tool for studying sirtuin biology.
A new probe for drug design: An affinity probe for Sirt2 has been developed that shows excellent selectivity and potency. The slow dissociation rate of the enzyme–ligand complex enables new applications, such as biolayer interferometry, and pull‐down assays for sirtuin rearranging ligands.
Reversible lysine deacetylation is exerted by both zinc and NAD+‐dependent deacetylases. It is an important factor in epigenetic regulation and more generally in the posttranslational regulation of ...protein stability, association and activity. Some of these enzymes can also cleave off fatty acids or dicarboxylic acids from lysines in proteins. The NAD+‐dependent deacetylases are termed Sirtuins and are implicated in the pathogenesis of different diseases. For the isotype Sirt2 highly selective inhibitors have been identified in the last few years. Many of those Sirt2 selective compounds, like the Sirtuin rearranging ligands (SirReals) discovered in our group, have been shown or are postulated to bind to the so‐called selectivity pocket. This binding site is not observed in crystal structures of the apo‐enzyme but can be opened up by long chain fatty acid substrates respectively suitable inhibitors. Recently, this unique feature of Sirt2 was exploited to provide highly potent and selective tools for the chemical biology of Sirtuins. Here, we shortly review Sirtuin biology, present inhibitors that have either been confirmed or postulated to bind to the selectivity pocket, their applications and an outlook regarding mechanistic investigations.
Sirtuins are NAD+dependent lysine deacetylases that can also cleave off larger acyl groups and dicarboxylic acids. They are postulated to be targets to treat cancer, neuro‐degeneration and metabolic diseases. Here, we review inhibitors of Sirt2 with a focus on ligands to the so‐called selectivity pocket, such as the SirReals (Sirtuin rearranging ligands).
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Ligands for the bromodomain and extra-terminal domain (BET) family of bromodomains have shown promise as useful therapeutic agents for treating a range of cancers and inflammation. ...Here we report that our previously developed 3,5-dimethylisoxazole-based BET bromodomain ligand (OXFBD02) inhibits interactions of BRD4(1) with the RelA subunit of NF-κB, in addition to histone H4. This ligand shows a promising profile in a screen of the NCI-60 panel but was rapidly metabolised (t½ = 39.8 min). Structure-guided optimisation of compound properties led to the development of the 3-pyridyl-derived OXFBD04. Molecular dynamics simulations assisted our understanding of the role played by an internal hydrogen bond in altering the affinity of this series of molecules for BRD4(1). OXFBD04 shows improved BRD4(1) affinity (IC50 = 166 nM), optimised physicochemical properties (LE = 0.43; LLE = 5.74; SFI = 5.96), and greater metabolic stability (t½ = 388 min).
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Lysine acetylation has emerged as a key post-translational modification found at many sites throughout the cell. It plays an important role in epigenetic processes, and more generally ...in the regulation of protein stability and interactions. Acetyl groups are installed by lysine acetyltransferases and removed by lysine deacetylases. Acetylated lysine residues function as binding sites for bromodomains, which are epigenetic reader protein modules that mediate protein–protein interactions. Progress in the development of small molecules that interfere with lysine acetylation has stimulated intensive research activity in diverse therapeutic areas. Some of these compounds are already marketed as drugs or are undergoing clinical trials. Here we review recent progress in the development of small molecules that interfere with lysine acetylation state and acetyl-lysine reading by bromodomains.
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