Design, Synthesis, and In Vivo Evaluation of Isosteviol Derivatives as New SIRT3 Activators with Highly Potent Cardioprotective Effects.

Cardiovascular diseases (CVDs) persist as the predominant cause of mortality, urging the exploration of innovative pharmaceuticals. Mitochondrial dysfunction stands as a pivotal contributor to CVDs development. Sirtuin 3 (SIRT3), a prominent mitochondrial deacetylase known for its crucial role in protecting mitochondria against damage and dysfunction, has emerged as a promising therapeutic target for CVDs treatment. Utilizing isosteviol, a natural ent-beyerene diterpenoid, 24 derivatives were synthesized and evaluated in vivo using a zebrafish model, establishing a deduced structure-activity relationship. Among these, derivative 5v exhibited significant efficacy in doxorubicin-induced cardiomyopathy in zebrafish and murine models. Subsequent investigations revealed that 5v selectively elevated SIRT3 expression, leading to the upregulation of SOD2 and OPA1 expression, effectively preventing mitochondrial dysfunction, mitigating oxidative stress, and preserving cardiomyocyte viability. As a novel structural class of SIRT3 activators with robust therapeutic effects, 5v emerges as a promising candidate for further drug development.

Glycyrrhizic acid exhibits strong anticancer activity in colorectal cancer cells via SIRT3 inhibition.

Sirtuin-3 (SIRT3) has been described as a colorectal cancer oncogene and to be regulated by glycyrrhizic acid (GA). However, few studies have explored the interaction between GA and SIRT3. Therefore, in the present study, we showed that GA could significantly decrease SIRT3 protein levels in SW620 and HT29 cells in a dose-dependent manner. Then, we overexpressed SIRT3 by lentivirus infection on SW620 and HT29 cells. We found that, in vitro, GA treatment significantly decreased cell viability, cell clone number, and invasion and migration number, besides significantly increasing apoptosis. Also, GA treatment significantly decreased the Bax/Bcl2 protein ratio and the expression of Cyclin D1, CDK2, CDK4, MMP-9, N-cadherin, and vimentin in SW620 and HT29 cells. Meanwhile, the SIRT3 overexpression could significantly reverse these changes. Moreover, the GA treatment could significantly decrease the weight of xenograft tumor tissues and its SIRT3 protein levels in vivo, while SIRT3 overexpression reversed these effects. Overall, GA inhibited the proliferation, invasion, and migration of colorectal cancer cells, and induced their apoptosis by SIRT3 inhibition.

Hydrogen Sulfide Attenuates Angiotensin II-Induced Cardiac Fibroblast Proliferation and Transverse Aortic Constriction-Induced Myocardial Fibrosis through Oxidative Stress Inhibition via Sirtuin 3.

Sirtuin 3 (SIRT3) is critical in mitochondrial function and oxidative stress. Our present study investigates whether hydrogen sulfide (H2S) attenuated myocardial fibrosis and explores the possible role of SIRT3 on the protective effects. Neonatal rat cardiac fibroblasts were pretreated with NaHS followed by angiotensin II (Ang II) stimulation. SIRT3 was knocked down with siRNA technology. SIRT3 promoter activity and expression, as well as mitochondrial function, were measured. Male wild-type (WT) and SIRT3 knockout (KO) mice were intraperitoneally injected with NaHS followed by transverse aortic constriction (TAC). Myocardium sections were stained with Sirius red. Hydroxyproline content, collagen I and collagen III, α-smooth muscle actin (α-SMA), and dynamin-related protein 1 (DRP1) expression were measured both in vitro and in vivo. We found that NaHS enhanced SIRT3 promoter activity and increased SIRT3 mRNA expression. NaHS inhibited cell proliferation and hydroxyproline secretion, decreased collagen I, collagen III, α-SMA, and DRP1 expression, alleviated oxidative stress, and improved mitochondrial respiration function and membrane potential in Ang II-stimulated cardiac fibroblasts, which were unavailable after SIRT3 was silenced. In vivo, NaHS reduced hydroxyproline content, ameliorated perivascular and interstitial collagen deposition, and inhibited collagen I, collagen III, and DRP1 expression in the myocardium of WT mice but not SIRT3 KO mice with TAC. Altogether, NaHS attenuated myocardial fibrosis through oxidative stress inhibition via a SIRT3-dependent manner.

Glucose limitation activates AMPK coupled SENP1-Sirt3 signalling in mitochondria for T cell memory development.

Metabolic programming and mitochondrial dynamics along with T cell differentiation affect T cell fate and memory development; however, how to control metabolic reprogramming and mitochondrial dynamics in T cell memory development is unclear. Here, we provide evidence that the SUMO protease SENP1 promotes T cell memory development via Sirt3 deSUMOylation. SENP1-Sirt3 signalling augments the deacetylase activity of Sirt3, promoting both OXPHOS and mitochondrial fusion. Mechanistically, SENP1 activates Sirt3 deacetylase activity in T cell mitochondria, leading to reduction of the acetylation of mitochondrial metalloprotease YME1L1. Consequently, deacetylation of YME1L1 suppresses its activity on OPA1 cleavage to facilitate mitochondrial fusion, which results in T cell survival and promotes T cell memory development. We also show that the glycolytic intermediate fructose-1,6-bisphosphate (FBP) as a negative regulator suppresses AMPK-mediated activation of the SENP1-Sirt3 axis and reduces memory development. Moreover, glucose limitation reduces FBP production and activates AMPK during T cell memory development. These data show that glucose limitation activates AMPK and the subsequent SENP1-Sirt3 signalling for T cell memory development.

Pharmacological Advantage of SIRT2-Selective versus pan-SIRT1-3 Inhibitors.

Because of their involvement in various biological pathways, the sirtuin enzyme family members SIRT1, SIRT2, and SIRT3 play both tumor-promoting and tumor-suppressing roles, based on the context and experimental conditions. Thus, an interesting question is whether inhibiting one of them or inhibiting all of them would be better for treating cancers. Pharmacologically, this is difficult to address, due in part to potential off-target effects of different compounds. Compounds with almost identical properties but differing in SIRT1-3 selectivity will be useful for addressing this question. Here, we have developed a pan SIRT1-3 inhibitor (NH4-6) and a SIRT2-selective inhibitor (NH4-13) with very similar chemical structures, with the only difference being the substitution of an ester bond to an amide bond. Such a minimal difference allows us to accurately compare the anticancer effect of pan SIRT1-3 inhibition and SIRT2-selective inhibition in cellular and mouse models. NH4-6 showed stronger cytotoxicity than NH4-13 in cancer cell lines. In mice, both inhibitors showed similar anticancer efficacy. However, NH4-6 is toxic to mice, which hinders the use of higher dosages. These results highlight the advantage of SIRT2-selective inhibitors as potential anticancer therapeutics.

Tubeimoside I protects against sepsis-induced cardiac dysfunction via SIRT3.

Sepsis-induced cardiac dysfunction (SICD) is one of the key complications in sepsis and it is associated with adverse outcomes and increased mortality. There is no effective drug to treat SICD. Previously, we reported that tubeimoside I (TBM) improved survival of septic mice. The aim of this study is to figure out whether TBM ameliorates SICD. Also, SIRT3 was reported to protects against SICD. Our second aim is to confirm whether SIRT3 plays essential roles in TBM's protective effects against SICD. Our results demonstrated that TBM could alleviate SICD and SICD's key pathological factor, inflammation, oxidative stress, and apoptosis were all reduced by TBM. Notably, SICD induced a significant decrease in cardiac SIRT3 expression, while TBM treatment could reverse SIRT3 expression. To clarify whether TBM provides protection via SIRT3, we injected a specific SIRT3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) into mice before TBM treatment. Then the cardioprotective effects of TBM were largely abolished by 3-TYP. This suggests that SIRT3 plays an essential role in TBM's cardioprotective effects. In vitro, TBM also protected H9c2 cells against LPS-induced injury, and siSIRT3 diminished these protective effects. Taken together, our results demonstrate that TBM protects against SICD via SIRT3. TBM might be a potential drug candidate for SICD treatment.

Inhibition of Sirtuin 3 prevents titanium particle-induced bone resorption and osteoclastsogenesis via suppressing ERK and JNK signaling.

Implant-derived wear particles can be phagocytosed by local macrophages, triggering an inflammatory cascade that can drive the activation and recruitment of osteoclasts, thereby inducing peri-prosthetic osteolysis. Efforts to suppress pro-inflammatory cytokine release and osteoclastsogenesis thus represent primary approaches to treating and preventing such osteolysis. Sirtuin 3 (SIRT3) is a NAD+-dependent deacetylases that control diverse metabolic processes. However, whether SIRT3 could mitigate wear debris-induced osteolysis has not been reported. Herein we explored the impact of the SIRT3 on titanium particle-induced osteolysis. Tartrate resistant acid phosphatase (TRAP) staining revealed that the inhibition of SIRT3 suppressed nuclear factor-κB ligand (RANKL)-mediated osteoclasts activation in a dose-dependent fashion. Notably, inhibition of SIRT3 also suppressed matrix metallopeptidase 9 (MMP9) and nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) expression at the mRNA and protein levels, while also inhibiting the mRNA expression of dendritic cell-specific transmembrane protein (DC-STAMP), ATPase H+ Transporting V0 Subunit D2 (Atp6v0d2), TRAP and Cathepsin K (CTSK) . In addition, inhibition of SIRT3 suppressed titanium particle-induced tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) expression and prevented titanium particle-induced osteolysis and bone loss in vivo. This inhibition of osteoclasts differentiation was found to be linked to the downregulation and reduced phosphorylation of JNK and ERK. Taken together, inhibition of SIRT3 may be a potential target for titanium particle-induced bone loss.

Discovery and characterization of small molecule SIRT3-specific inhibitors as revealed by mass spectrometry.

Sirtuins play a prominent role in several cellular processes and are implicated in various diseases. The understanding of biological roles of sirtuins is limited because of the non-availability of small molecule inhibitors, particularly the specific inhibitors directed against a particular SIRT. We performed a high-throughput screening of pharmacologically active compounds to discover novel, specific, and selective sirtuin inhibitor. Several unique in vitro sirtuin inhibitor pharmacophores were discovered. Here, we present the discovery of novel chemical scaffolds specific for SIRT3. We have demonstrated the in vitro activity of these compounds using label-free mass spectroscopy. We have further validated our results using biochemical, biophysical, and computational studies. Determination of kinetic parameters shows that the SIRT3 specific inhibitors have a moderately longer residence time, possibly implying high in vivo efficacy. The molecular docking results revealed the differential selectivity pattern of these inhibitors against sirtuins. The discovery of specific inhibitors will improve the understanding of ligand selectivity in sirtuins, and the binding mechanism as revealed by docking studies can be further exploited for discovering selective and potent ligands targeting sirtuins.

Nutraceutical based SIRT3 activators as therapeutic targets in Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease, and its incidence is increasing worldwide with increased lifespan. Currently, there is no effective treatment to cure or prevent the progression of AD, which indicates the need to develop novel therapeutic targets and agents. Sirtuins, especially SIRT3, a mitochondrial deacetylase, are NAD-dependent histone deacetylases involved in aging and longevity. Accumulating evidence indicates that SIRT3 dysfunction is strongly associated with pathologies of AD, hence, therapeutic modulation of SIRT3 activity may be a novel application to ameliorate the pathologies of AD. Natural products commonly used in traditional medicine have wide utility and appear to have therapeutic benefits for the treatment of neurodegenerative diseases such as AD. The present review summarizes the currently available natural SIRT3 activators and their potentially neuroprotective molecular mechanisms of action that make them a promising agent in the treatment and management of neurodegenerative diseases such as AD.

Hit-to-lead optimization on aryloxybenzamide derivative virtual screening hit against SIRT.

Sirtuins (SIRTs) are a class of nicotinamide adenine dinucleotide (NAD+)-dependent protein histone deacetylases (HDACs) that are evolutionarily conserved from bacteria to mammals. This group of enzymes catalyses the reversible deacetylation of lysine residues in the histones or non-histone substrates using NAD+ as a cosubstrate. Numerous studies have demonstrated that the aberrant enzymatic activity of SIRTs has been linked to various diseases like diabetes, cancer, and neurodegenerative disorders. Previously, we performed a pharmacophore-based virtual screening campaign and an aryloxybenzamide derivative (1) displaying SIRT1/2 inhibitory effect was identified as a hit compound. In the current study, the hit-to-lead optimization on the hit compound was explored in order to improve the SIRT binding and inhibition. Fourteen compounds, ten of which were new, have been synthesized and subjected to in vitro biological evaluation for their inhibitory activity against SIRT1-3. By the structural modifications performed, a significant improvement was observed in selective SIRT1 inhibition for ST01, ST02, and ST11 compared to that of the hit compound. The highest SIRT2 inhibitory activity was observed for ST14, which was designed according to compatibility with pharmacophore model developed for SIRT2 inhibitors and thus, providing the interactions required with key residues in SIRT2 active site. Furthermore, ST01, ST02, ST11, and ST14 were subjected to in vitro cytotoxicity assay against MCF-7 human breast cancer cell line to determine the influence of the improvement in SIRT1/2 inhibition along with the structural modifications on the cytotoxic properties of the compounds. The cytotoxicity of the compounds was found to be correlated with their SIRT inhibitory profiles indicating the effects of SIRT1/2 inhibition on cancer cell viability. Overall, this study provides structural insights for further inhibitor improvement.

Sirt3 regulates adipogenesis and adipokine secretion via its enzymatic activity.

The purpose of this research was to identify if Sirt3 plays a role in marrow adipogenesis and adipokines secretion, especially adiponectin using bone marrow-derived stroma (ST2) cell model. Sirt3 overexpression leads to a significant increase in adipogenesis compared to controls. The induction of adipogenesis by Sirt3 is associated with increased gene expression of adipocyte markers as well as adiponectin/adipokines. In sharp contrast, the inhibition of Sirt3 exhibited significantly decreased adipogenesis, adipocyte markers, and adiponectin/adipokines compared to the controls. Interestingly, perilipin 1 (Plin 1) expression was decreased in Sirt3 induction but increased in Sirt3 inhibition. One hundred and fifteen mitochondrial acetylated peptides from 67 mitochondrial proteins had lower levels of acetylation in adipocytes induced by Sirt3 overexpression (Sirt3OE) compared to the control. Of the 67 proteins less enriched in acetylation, 22 acetylated proteins were decreased by more than twofold. These proteins are considered potential Sirt3 substrates in adipogenesis. In conclusion, Sirt3 has a novel, important role in modulating adipogenesis and adiponectin/adipokine expression. The connection axis among Sirt3-adipogenesis-adipokines was linked to its substrates by mass spectrometry analysis. These findings contribute to the efforts of revealing Sirt3 functions and Sirt3 usage as a potential target for treatment of metabolic homeostasis and diseases including type 2 diabetes.

LCZ696 Ameliorates Oxidative Stress and Pressure Overload-Induced Pathological Cardiac Remodeling by Regulating the Sirt3/MnSOD Pathway.

AIMS: We aimed to investigate whether LCZ696 protects against pathological cardiac hypertrophy by regulating the Sirt3/MnSOD pathway. METHODS: In vivo, we established a transverse aortic constriction animal model to establish pressure overload-induced heart failure. Subsequently, the mice were given LCZ696 by oral gavage for 4 weeks. After that, the mice underwent transthoracic echocardiography before they were sacrificed. In vitro, we introduced phenylephrine to prime neonatal rat cardiomyocytes and small-interfering RNA to knock down Sirt3 expression. RESULTS: Pathological hypertrophic stimuli caused cardiac hypertrophy and fibrosis and reduced the expression levels of Sirt3 and MnSOD. LCZ696 alleviated the accumulation of oxidative reactive oxygen species (ROS) and cardiomyocyte apoptosis. Furthermore, Sirt3 deficiency abolished the protective effect of LCZ696 on cardiomyocyte hypertrophy, indicating that LCZ696 induced the upregulation of MnSOD and phosphorylation of AMPK through a Sirt3-dependent pathway. CONCLUSIONS: LCZ696 may mitigate myocardium oxidative stress and apoptosis in pressure overload-induced heart failure by regulating the Sirt3/MnSOD pathway.

Sirtuin 3 Inhibits Airway Epithelial Mitochondrial Oxidative Stress in Cigarette Smoke-Induced COPD.

Mitochondrial damage in airway epithelial cells plays an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). Sirtuin 3 (Sirt3) is a mitochondrial deacetylase regulating mitochondrial function, but its role in the pathogenesis of COPD is still unknown. The aim of the present study was to investigate the effect of Sirt3 on airway epithelial mitochondria in cigarette smoke-induced COPD. Our present study has shown serious airway inflammation, alveolar space enlargement, and mitochondrial damage of the airway epithelium in COPD rats. Compared to the control rats, Sirt3 protein expression was significantly decreased in the airway epithelium and lung tissue homogenate from COPD rats. In airway epithelial cells (BEAS-2B), cigarette smoke extract (CSE) treatment significantly decreased mRNA and protein expression of Sirt3 and manganese superoxide dismutase (MnSOD), as well as MnSOD activity in a concentration and time-dependent manner. Sirt3 siRNA further significantly intensified the decreases in MnSOD expression and activity and aggravated mitochondrial oxidative stress and cell injury when airway epithelial cells were treated with 7.5% CSE. In contrast, Sirt3 overexpression significantly prevented the decrease of MnSOD expression and activity and improved mitochondrial oxidative stress and cell injury in CSE-treated airway epithelial cells. These data suggest that Sirt3 inhibits airway epithelial mitochondrial oxidative stress possibly through the regulation of MnSOD, thereby contributing to the pathogenesis of COPD.

Oleanolic Acid Decreases IL-1ß-Induced Activation of Fibroblast-Like Synoviocytes via the SIRT3-NF-κB Axis in Osteoarthritis.

Synovial inflammation is a major pathological feature of osteoarthritis (OA), which is a chronic degenerative joint disease. Fibroblast-like synoviocytes (FLS), localized in the synovial membrane, are specialized secretory cells. During OA synovitis, FLS produce chemokines and cytokines that stimulate chondrocytes to secrete inflammatory cytokines and activate matrix metalloproteinases (MMPs) in FLS. Recent studies have demonstrated that sirtuin 3 (SIRT3) performs as a key regulator in maintaining mitochondrial homeostasis in OA. This study aims at ascertaining whether SIRT3 is involved in OA synovitis. The overexpression (OE) and knockdown (KD) of SIRT3 are established by short hairpin RNA (shRNA) and recombinant plasmid in human FLS. The anti-inflammatory effect of SIRT3 underlying in oleanolic acid- (OLA-) prevented interleukin-1ß- (IL-1ß-) induced FLS dysfunction is then evaluated in vitro. Additionally, the molecular mechanisms of SIRT3 are assessed, and the interaction between SIRT3 and NF-κB is investigated. The data suggested that SIRT3 can be detected in human synovial tissues during OA, and OLA could elevate SIRT3 expression. OE-SIRT3 and OLA exhibited equal authenticity to repress inflammation and reverse oxidative stress changes in IL-1ß-induced human FLS dysfunction. KD-SIRT3 was found to exacerbate inflammation and oxidative stress changes in human FLS. Furthermore, it was found that SIRT3 could directly bind with NF-κB, resulting in the suppression of NF-κB activation induced by IL-1ß in human FLS, which then repressed synovial inflammation in OA. In general, the activation of SIRT3 by OLA inhibited synovial inflammation by suppressing the NF-κB signal pathway in FLS, and this suggested that SIRT3 is a potential target for OA synovitis therapy.

Withaferin A Exerts Preventive Effect on Liver Fibrosis through Oxidative Stress Inhibition in a Sirtuin 3-Dependent Manner.

Sirtuin 3 (SIRT3) is a deacetylase involved in the development of many inflammation-related diseases including liver fibrosis. Withaferin A (WFA) is a bioactive constituent derived from the Withania somnifera plant, which has extensive pharmacological activities; however, little is known about the regulatory role of SIRT3 in the WFA-induced antifibrogenic effect. The current study is aimed at investigating the role of SIRT3 in WFA-induced antioxidant effects in liver fibrosis. Our study verified that WFA attenuated platelet-derived growth factor BB- (PDGF-BB-) induced liver fibrosis and promoted PDGF-BB-induced SIRT3 activity and expression in JS1 cells. SIRT3 silencing attenuated the antifibrogenic and antioxidant effects of WFA in activated JS1 cells. Moreover, WFA inhibited carbon tetrachloride- (CCl4-) induced liver injury, collagen deposition, and fibrosis; increased the SIRT3 expression; and suppressed the CCl4-induced oxidative stress in fibrotic livers of C57/BL6 mice. Furthermore, the antifibrogenic and antioxidant effects of WFA could be available in CCl4-induced WT (129S1/SvImJ) mice but were unavailable in CCl4-induced SIRT3 knockout (KO) mice. Our study suggested that WFA inhibited liver fibrosis through the inhibition of oxidative stress in a SIRT3-dependent manner. WFA could be a potential compound for the treatment of liver fibrosis.

Dioscin Protects against Aß1-42 Oligomers-Induced Neurotoxicity via the Function of SIRT3 and Autophagy.

Alzheimer's disease (AD) is a common neurodegenerative disease with high incidence among old people. Dioscin is a product extracted from natural herbs, which has multiple pharmacological activities. In this study, we investigated the potential effects of disocin on amyloid-ß peptide (Aß1-42) oligomers-treated HT22 cells. Aß1-42 oligomers induced great neurotoxicity to HT22 cells as examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results of terminal deoxynucleoitidyl transferase-mediated deoxyuridine triphosphate biotin nich end labeling (TUNEL) staining and flow cytometry indicated that Aß1-42 oligomers led to increased apoptosis and generation of reactive oxygen species (ROS) in HT22 cells. However, dioscin could remarkably inhibit the neurotoxicity induced by Aß1-42 oligomers, as well as decrease the apoptosis and ROS generation. Sirtuin-3 (SIRT3) staining and quantification indicated that dioscin upregulated the expression of neuroprotective SIRT3. Moreover, dioscin induced the formation of autophagosomes and autolysosomes in HT22 cells. Dioscin also enhanced the levels of Beclin-1 and LC3-II while decreased the level of p62. These results suggested that dioscin could activate autophagy in HT22 cells. It was also found that knocking down SIRT3 resulted in the downregulation of Beclin-1, LC3-II and the aggregation of p62, suggesting that SIRT3 was an important regulator in autophagy. Furthermore, we found that knocking down SIRT3 or inhibiting autophagy suppressed the protective effects of dioscin on Aß1-42 oligomers-induced neurotoxicity, apoptosis and ROS generation. These results revealed that SIRT3 and autophagy functioned together in the neuroprotective mechanisms of dioscin. Therefore, dioscin might be a promising drug to protect against Aß1-42 oligomers-induced neurotoxicity and reduce neuron damage or death in AD.

Mitochondrial Sirtuin 3: New emerging biological function and therapeutic target.

Sirtuin 3 (SIRT3) is one of the most prominent deacetylases that can regulate acetylation levels in mitochondria, which are essential for eukaryotic life and inextricably linked to the metabolism of multiple organs. Hitherto, SIRT3 has been substantiated to be involved in almost all aspects of mitochondrial metabolism and homeostasis, protecting mitochondria from a variety of damage. Accumulating evidence has recently documented that SIRT3 is associated with many types of human diseases, including age-related diseases, cancer, heart disease and metabolic diseases, indicating that SIRT3 can be a potential therapeutic target. Here we focus on summarizing the intricate mechanisms of SIRT3 in human diseases, and recent notable advances in the field of small-molecule activators or inhibitors targeting SIRT3 as well as their potential therapeutic applications for future drug discovery.

Melatonin Attenuates Anoxia/Reoxygenation Injury by Inhibiting Excessive Mitophagy Through the MT2/SIRT3/FoxO3a Signaling Pathway in H9c2 Cells.

PURPOSE: Autophagy caused by ischemia/reperfusion (I/R) increases the extent of cardiomyocyte damage. Melatonin (Mel) diminishes cardiac injury through regulating autophagy and mitochondrial dynamics. However, illustrating the specific role of mitophagy in the cardioprotective effects of melatonin remains a challenge. The aim of our research was to investigate the impact and underlying mechanisms of melatonin in connection with mitophagy during anoxia/reoxygenation (A/R) injury in H9c2 cells. METHODS: H9c2 cells were pretreated with melatonin with or without the melatonin membrane receptor 2 (MT2) antagonist 4-P-PDOT, the MT2 agonist IIK7 and the sirtuin 3 (SIRT3) inhibitor 3-TYP for 4 hours and then subjected to A/R injury. Cell viability, cellular apoptosis, necrosis levels and oxidative markers were assessed. The expression of SIRT3 and forkhead box O3a (FoxO3a), mitochondrial function and the levels of mitophagy-related proteins were also evaluated. RESULTS: A/R injury provoked enhanced mitophagy in H9c2 myocytes. In addition, increased mitophagy was correlated with decreased cellular viability, increased oxidative stress and mitochondrial dysfunction in H9c2 cells. However, melatonin pretreatment notably increased cell survival and decreased cell apoptosis and oxidative response after A/R injury, accompanied by restored mitochondrial function. The inhibition of excessive mitophagy is involved in the cardioprotective effects of melatonin, as shown by the decreased expression of the mitophagy-related molecules Parkin, Beclin1, and BCL2-interacting protein 3-like (BNIP3L, best known as NIX) and decreased light chain 3 II/light chain 3 I (LC3 II/LC3 I) ratio and upregulation of p62 expression. Moreover, the decreased expression of SIRT3 and FoxO3a in A/R-injured H9c2 cells was abrogated by melatonin, but these beneficial effects were attenuated by the MT2 antagonist 4-P-PDOT or the SIRT3 inhibitor 3-TYP and enhanced by the MT2 agonist IIK7. CONCLUSION: These results indicate that melatonin protects H9c2 cells during A/R injury through suppressing excessive mitophagy by activating the MT2/SIRT3/FoxO3a pathway. Melatonin may be a useful candidate for alleviating myocardial ischemia/reperfusion (MI/R) injury in the future, and the MT2 receptor might become a therapeutic target.

Design, Synthesis, and Biological Evaluation of 8-Mercapto-3,7-Dihydro-1H-Purine-2,6-Diones as Potent Inhibitors of SIRT1, SIRT2, SIRT3, and SIRT5.

Sirtuins (SIRT1-7) are a family of NAD+-dependent deacetylases. They regulate many physiological processes and play important roles in inflammation, diabetes, cancers, and neurodegeneration diseases. Sirtuin inhibitors have potential applications in the treatment of neurodegenerative diseases and various cancers. Herein, we identified new sirtuin inhibitors based on the scaffold of 8-mercapto-3,7-dihydro-1H-purine-2,6-dione. To elucidate the inhibitory mechanism, the binding modes of the inhibitors in SIRT3 were established by molecular docking, showing that the inhibitors occupy the acetyl lysine binding site and interact with SIRT3, mainly through hydrophobic interactions. The interactions were validated by site-directed mutagenesis of SIRT3 and structure-activity relationship analysis of the inhibitors. Consistently, enzyme kinetic assays and microscale thermophoresis showed that these compounds are competitive inhibitors to the acetyl substrate, and mix-type inhibitors to NAD+. Furthermore, we demonstrated that the compounds are potent SIRT1/2/3/5 pan-inhibitors. This study provides novel hits for developing more potent sirtuin inhibitors.

Inhibition of the Peroxisome Proliferator-Activated Receptor gamma Coactivator 1-alpha (PGC-1α)/Sirtuin 3 (SIRT3) Pathway Aggravates Oxidative Stress After Experimental Subarachnoid Hemorrhage.

BACKGROUND Emerging evidence shows that Sirtuin 3 (SIRT3) can exert an antioxidative effect in various neurodegenerative diseases, but whether and how SIRT3 modulates neuronal death after subarachnoid hemorrhage (SAH) remains to be elucidated. MATERIAL AND METHODS Experimental SAH was induced in adult mice by prechiasmatic cistern injection and primary neurons by OxyHb incubation. The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1alpha) and SIRT3 protein levels were examined at different time points after SAH induction. The PGC-1alpha protein gene knockdown in vivo and in vitro was achieved by transfection of lentivirus (LV) vectors expressing shPGC-1alpha or negative control (NC). Western blot, oxidative stress index, histopathology, neurological function, and cell viability analysis was performed. RESULTS Results showed that the PGC-1alpha/SIRT3 pathway was remarkably activated in vivo and in vitro after SAH. LV-shPGC-1alpha treatment significantly inhibited the activation of this pathway after SAH, accompanied by deteriorated neurologic function, aggravated oxidative stress, increased neuronal apoptosis, and enhanced cytotoxicity compared with the mice or primary neurons treated with LV-NC only. CONCLUSIONS The present results highlight the detrimental PGC-1alpha/SIRT3 pathway, involving regulation of the endogenous antioxidant activity against neuronal damage, which may provide a potential therapeutic target in SAH.