SirReal2

Target: Sirtuin 2 (Sirt2)

IC50: 140 nM

SirReal2 is a potent and selective Sirtuin 2 (Sirt2) inhibitor with IC50 value of 140 nM [1]. The Sirtuins are a highly conserved class of NAD+-dependent lysine deacylases. The human isotype Sirtuin 2 (Sirt2) deacetylates both cytoplasmatic and nuclear proteins and it has been implicated in the pathogenesis of cancer, neurodegeneration, and inflammation [1, 2]. Therefore, the modulation of Sirt2 activity is a promising strategy for pharmaceutical intervention.

In vitro: SirReal2, the most potent Sirtuin-rearranging ligand, induced the checkpoint protein BubR1 destabilization and hyperacetylation of the microtubule network in HeLa cells [1]. Moreover, SirReal2 (20 μM) inhibited Sirt2 activity but was unable to affect the activity of the other Class-I sirtuins Sirt1 and Sirt3 in cells. SirReal2 (12.5, 25, and 50 μM) treatment dose-dependently induced depletion of BubR1 but did not alter cell cycle distribution [1].

In vivo: SirReal2 (12.5, 25, and 50 μM) treatment did not induce the increase in acetylation of p53 [1].

References:
1.  Rumpf T, Schiedel M, Karaman B, Roessler C, North BJ, Lehotzky A, et al. Selective Sirt2 inhibition by ligand-induced rearrangement of the active site. Nat Commun. 2015;6:6263. Epub 2015/02/13.
2.  Galleano I, Schiedel M, Jung M, Madsen AS, Olsen CA. A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluation. J Med Chem. 2016;59(3):1021-31. Epub 2016/01/21.

SIRT2 Inhibition Results in Meiotic Arrest, Mitochondrial Dysfunction, and Disturbance of Redox Homeostasis during Bovine Oocyte Maturation.[Pubmed:30889926]

Int J Mol Sci. 2019 Mar 18;20(6). pii: ijms20061365.

SIRT2, a member of the sirtuin family, has been recently shown to exert important effects on mitosis and/or metabolism. However, its roles in oocyte maturation have not been fully clarified. In this study, SIRT2, located in the cytoplasm and nucleus, was found in abundance in the meiotic stage, and its expression gradually decreased until the blastocyst stage. Treatment with SIRT2 inhibitors resulted in the prevention of oocyte maturation and the formation of poor-quality oocytes. By performing confocal scanning and quantitative analysis, the results showed that SIRT2 inhibition induced prominent defects in spindle/chromosome morphology, and led to the hyperacetylation of alpha-tubulin and H4K16. In particular, SIRT2 inhibition impeded cytoplasmic maturation by disturbing the normal distribution of cortical granules, endoplasmic reticulum, and mitochondria during oocyte meiosis. Meanwhile, exposure to SirReal2 led to elevated intracellular reactive oxygen species (ROS) accumulation, low ATP production, and reduced mitochondrial membrane potential in oocytes. Further analysis revealed that SIRT2 inhibition modulated mitochondrial biogenesis and dynamics via the downregulation of TFAM and Mfn2, and the upregulation of DRP1. Mechanistically, SIRT2 inhibition blocked the nuclear translocation of FoxO3a by increasing FoxO3a acetylation, thereby downregulating the expression of FoxO3a-dependent antioxidant genes SOD2 and Cat. These results provide insights into the potential mechanisms by which SIRT2-dependent deacetylation activity exerts its effects on oocyte quality.

Direct Comparison of SIRT2 Inhibitors: Potency, Specificity, Activity-Dependent Inhibition, and On-Target Anticancer Activities.[Pubmed:30058233]

ChemMedChem. 2018 Sep 19;13(18):1890-1894.

Sirtuin inhibitors have attracted much interest due to the involvement of sirtuins in various biological processes. Several SIRT2-selective inhibitors have been developed, and some exhibit anticancer activities. To facilitate the choice of inhibitors in future studies and the development of better inhibitors, we directly compared several reported SIRT2-selective inhibitors: AGK2, SirReal2, Tenovin-6, and TM. In vitro, TM is the most potent and selective inhibitor, and only TM could inhibit the demyristoylation activity of SIRT2. SirReal2, Tenovin-6, and TM all showed cytotoxicity in cancer cell lines, with Tenovin-6 being the most potent, but only TM showed cancer-cell-specific toxicity. All four compounds inhibited the anchorage-independent growth of HCT116 cells, but the effect of TM was most significantly affected by SIRT2 overexpression, suggesting that the anticancer effect of TM depends more on SIRT2 inhibition. These results not only provide useful guidance about choosing the right SIRT2 inhibitor in future studies, but also suggest general practices that should be followed for small-molecule inhibitor development activities.

SIRT2 plays a novel role on progesterone, estradiol and testosterone synthesis via PPARs/LXRalpha pathways in bovine ovarian granular cells.[Pubmed:30009951]

J Steroid Biochem Mol Biol. 2019 Jan;185:27-38.

SIRT2 has been shown to possess NAD(+)-dependent deacetylase and desuccinylase enzymatic activities, it also regulates metabolism homeostasis in mammals. Previous data has suggested that resveratrol, a potential activator of Sirtuins, played a stimulation role in steroidogenesis. Unfortunately, to date, the physiological roles of SIRT2 in ovarian granular cells (GCs) are largely unknown. Here, we studied the function and molecular mechanisms of SIRT2 on steroid hormone synthesis in GCs from Qinchuan cattle. Immunohistochemistry and western blotting showed that SIRT2 was expressed not only in GCs and cumulus cells, but also in oocytes and theca cells. We found that the secretion of progesterone was induced, whereas that of estrogen and testosterone secretion was suppressed by treatment with the SIRT2 inhibitor (Thiomyristoyl or SirReal2) or siRNA. Additionally, the PPARs/LXRalpha signaling pathways were suppressed by SIRT2 siRNA or inhibitors. The mRNA expression of CYP17, aromatase and StAR was suppressed, but the abundance of CYP11A1 mRNA was induced by SIRT2 inhibition. Furthermore, the PPARalpha agonist or PPARgamma antagonist could mimic the effects of SIRT2 inhibition on hormones levels and gene expression associated with steroid hormone biosynthesis. In turn, those effects were abolished by the LXRalpha agonist (LXR-623). Together, these data support the hypothesis that SIRT2 regulates steroid hormone synthesis via the PPARs/LXRalpha pathways in GCs.

SIRT2 Promotes the Migration and Invasion of Gastric Cancer through RAS/ERK/JNK/MMP-9 Pathway by Increasing PEPCK1-Related Metabolism.[Pubmed:29925042]

Neoplasia. 2018 Jul;20(7):745-756.

Metastasis is the most important feature of gastric cancer (GC) and the most widely recognized reason for GC-related deaths. Unfortunately, the underlying mechanism behind this metastasis remains unknown. Mounting evidence suggests the dynamic regulatory role of sirtuin2 (SIRT2), a histone deacetylase (HDAC), in cell migration and invasion. The present study aims to evaluate the biological function of SIRT2 in GC and identify the target of SIRT2 as well as evaluate its therapeutic efficacy. We found that SIRT2 was upregulated in GC tissues compared to adjacent normal tissues, and this was correlated with reduced patient survival. Although CCK8 and colony-formation assays showed that SIRT2 overexpression marginally promoted proliferation in GC cell lines, SIRT2 knockdown or treatment with SirReal2 decreased the migration and invasion of GC cells. We demonstrated both in vitro and in vivo that SirReal2 could inhibit the deacetylation activity of SIRT2 and its downstream target PEPCK1, which is related to mitochondrial metabolism and RAS/ERK/JNK/MMP-9 pathway. Taken together, these results demonstrate for the first time that SirReal2 selectively targets SIRT2 and decreases migration as well as invasion in human GC cells. SirReal2 therefore shows promise as a new drug candidate for GC therapy.

A Continuous, Fluorogenic Sirtuin 2 Deacylase Assay: Substrate Screening and Inhibitor Evaluation.[Pubmed:26788965]

J Med Chem. 2016 Feb 11;59(3):1021-31.

Sirtuins are important regulators of lysine acylation, which is implicated in cellular metabolism and transcriptional control. This makes the sirtuin class of enzymes interesting targets for development of small molecule probes with pharmaceutical potential. To achieve detailed profiling and kinetic insight regarding sirtuin inhibitors, it is important to have access to efficient assays. In this work, we report readily synthesized fluorogenic substrates enabling enzyme-economical evaluation of SIRT2 inhibitors in a continuous assay format as well as evaluation of the properties of SIRT2 as a long chain deacylase enzyme. Novel enzymatic activities of SIRT2 were thus established in vitro, which warrant further investigation, and two known inhibitors, suramin and SirReal2, were profiled against substrates containing epsilon-N-acyllysine modifications of varying length.

Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.[Pubmed:25672491]

Nat Commun. 2015 Feb 12;6:6263.

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.

SirReal2 is a potent, isotype-selective Sirt2 inhibitor with an IC50 value of 140 nM and has very little effect on the activities of Sirt3-5. SirReal2 leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1.

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