Design, synthesis and biological evaluation of prostate-specific membrane antigen (PSMA)-targeted SIRT2 inhibitors.

Sirtuins belong to a specific class of enzymes called NAD+-dependent protein deacetylases. Among them, SIRT2 is predominantly localized in the cytoplasm and plays a vital role in tumor development and progression. As a result, it becomes an important target for the development of anticancer drugs. While SIRT2 inhibitors have shown broad-spectrum cytotoxicity against various cancer cells, their ability to inhibit the growth of certain cancers like prostate cancer has been limited, possibly due to insufficient targeting properties. To overcome this limitation, our goal was to target prostate-specific membrane antigen (PSMA), a valuable biomarker for prostate cancer, using lysine-urea-glutamic acid (KUE) as a PSMA ligand. This approach allowed us to systematically design new SIRT2 inhibitors. Evaluation showed that compound 17 exhibited superior inhibitory activity, improved targeting properties, and enhanced antiproliferative efficacy specifically in prostate cancer cells. These findings suggest a promising strategy for utilizing SIRT2 inhibitors in prostate cancer therapy.

Effect of Bone Marrow Stromal Cells Derived miR-26b on Chondrocytes of Osteoporosis Rats.

OBJECTIVE: Osteoporosis is a common bone disease. miR-26b regulates OA-induced osteogenesis and induces osteoporosis. miR-26b is elevated in bone marrow stromal cells (BMSCs) during bone formation; however, we haven't fully revealed whether it is directly involved in this process, which was the aim of this study. METHODS: An oophorectomized rat model of osteoporosis was used. BMSCs were detected by electron microscopy of exosomes, and mir-26b levels were detected by RT-PCR. The correlation between mir-26b and sirt2 was detected by bioinformatics and luciferase activity analysis. Bone microstructure and cartilage moisture content were also measured. The proliferation ability of mir-26b and sirt2 on chondrocytes was detected by cell viability test and flow cytometry. RESULTS: Western blotting further proved that the surface markers of isolated granular exosomes were positive for CD63 and CD81. Further analysis showed that exosomes' diameters ranged from 50 to 150 nm. Mir-26b is elevated in BMSC, and its mimics can promote proliferation. Luciferase showed that mir-26b targets sirt2 and the effect of elevated mir-26b on chondrocytes was completely reversed by silencing sirt2. The proliferation ability of C28/I2 chondrocytes in Mir MICs group was lower than other two groups, while that in Mir inhibition group had stronger proliferation ability than in the Mir NC group. mir-26b was highly expressed in BMSC, indicating that mir-26b comes from secretion of BMSC. CONCLUSION: Mir-26 is highly expressed in OP. mir-26b can therefore target sirt2 to promote proliferation and inhibit apoptosis of OP chondrocytes. It may offer a possibility of a treatment of OP in the future.

Palmitic acid causes hepatocyte inflammation by suppressing the BMAL1-NAD+-SIRT2 axis.

Palmitic acid is the most abundant saturated fatty acid in circulation and causes hepatocyte toxicity and inflammation. As saturated fatty acid can also disrupt the circadian rhythm, the present work evaluated the connection between clock genes and NAD+ dependent Sirtuins in protecting hepatocytes from lipid-induced damage. Hepatocytes (immortal cells PH5CH8, hepatoma cells HepG2) treated with higher doses of palmitic acid (400-600µM) showed typical features of steatosis accompanied with growth inhibition and increased level of inflammatory markers (IL-6 IL-8, IL-1α and IL-1ß) together with decline in NAD+ levels. Palmitic acid treated hepatocytes showed significant decline in not only the protein levels of SIRT2 but also its activity as revealed by the acetylation status of its downstream targets (Tubulin and NF-ƙB). Additionally, the circadian expression of both SIRT2 and BMAL1 was inhibited in presence of palmitic acid in only the non-cancerous hepatocytes, PH5CH8 cells. Clinical specimens obtained from subjects with NASH-associated fibrosis, ranging from absent (F0) to cirrhosis (F4), showed a significant decline in levels of SIRT2 and BMAL1, especially in the cirrhotic liver. Ectopic expression of BMAL1 or activating SIRT2 by supplementation with nicotinamide riboside (precursor of NAD+) dampened the palmitic acid induced lipoinflammation and lipotoxicity more effectively in PH5CH8 cells as compared to HepG2 cells. Mechanistically, palmitic acid caused transcriptional suppression of SIRT2 by disrupting the chromatin occupancy of BMAL1 at its promoter site. Overall, the work suggested that SIRT2 is a clock-controlled gene that is transcriptionally regulated by BMAL1. In conclusion the activation of the BMAL1-NAD+-SIRT2 axis shows hepatoprotective effects by preventing lipotoxicity and dampening inflammation.

Calenduloside E Ameliorates Inflammatory Responses in Adipose Tissue via Sirtuin 2-NLRP3 Inflammasome Axis.

Obesity-related metabolic diseases are associated with a chronic inflammatory state. Calenduloside E (CE) is a triterpene saponin from sugar beet. In mouse models, CE reduced pro-inflammatory cytokines in white adipose tissue (WAT) and decreased macrophage infiltration of WAT. And CE inhibited pyroptosis in J774A.1 cells and WAT by inhibiting the activation of the nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome. Moreover, CE could trigger the activation of Sirtuin 2 (SIRT2), leading to a decrease in the acetylation of NLRP3, particularly at the K24 site. In addition, it has been shown that CE can reduce inflammation in adipocytes that have been induced by macrophage-conditioned medium. However, the selective SIRT2 inhibitor AGK2 hindered the beneficial effects of CE. In summary, CE has the capacity to impede NLRP3-mediated pyroptosis by triggering SIRT2 activity, thus positioning CE as a promising therapeutic avenue for combating obesity-related metabolic disorders.

Dendrobium nobile-derived polysaccharides stimulate the glycolytic pathway by activating SIRT2 to regulate insulin resistance in polycystic ovary syndrome granulosa cells.

Insulin resistance (IR) is one of the major complications of polycystic ovary syndrome (PCOS). This study aimed to investigate the effects and the molecular regulatory mechanism by which Dendrobium nobile-derived polysaccharides (DNP) improve IR in rats with letrozole and high-fat-diet induced PCOS. In vivo, DNP (200 mg/kg/d) administration not only reduced body weight, blood glucose, and insulin levels in PCOS rats, but also improve the disrupted estrous cycle. In addition, DNP treatment reduced atretic and cystic follicles and enhanced granulosa cell layer thickness, thereby restoring follicle development. In vitro, DNP treatment (100 µM) increased lactate levels and decreased pyruvate levels in insulin-treated (8 µg/mL) KGN cells. Additionally, DNP also decreased the expression of IGF1 and increased that of IGF1R, SIRT2, LDHA, PKM2 and HK2 both in vivo and in vitro. Also, SIRT2 expression was specifically inhibited by AGK2, while DNP significantly improved IR and glycolysis by reversing the effect of AGK2 treatment on lactate and pyruvate production, upregulating the expression levels of IGF1R, LDHA, HK2, and PKM2 and downregulating the expression level of IGF1. The results indicate that DNP can effectively improve IR and restore glycolytic pathway by activating SIRT2, which may provide a potential therapeutic approach for PCOS patients.

NMN partially rescues cuproptosis by upregulating sirt2 to increase intracellular NADPH.

Cuproptosis is characterized by lipoylated protein aggregation and loss of iron-sulfur (Fe-S) proteins, which are crucial for a wide range of important cellular functions, including DNA replication and damage repair. Sirt2 and sirt4 are lipoamidases that remove the lipoyl moiety from lipoylated proteins using nicotinamide adenine dinucleotide (NAD+) as a cofactor. However, to date, it is not clear whether nicotinamide mononucleotide (NMN), a precursor of NAD+, affects cellular sensitivity to cuproptosis. Therefore, in the current study, cuproptosis was induced by the copper (Cu) ionophore elesclomol (Es) in HeLa cells. It was also found that Es/Cu treatment increased cellular DNA damage level. On the other hand, NMN treatment partially rescued cuproptosis in a dose-dependent manner, as well as reduced cellular DNA damage level. In addition, NMN upregulated the expression of Fe-S protein POLD1, without affecting the aggregation of lipoylated proteins. Mechanistic study revealed that NMN increased the expression of sirt2 and cellular reduced nicotinamide adenine dinucleotide phosphate (NADPH) level. Overexpression of sirt2 and sirt4 did not change the aggregation of lipoylated proteins, however, sirt2, but not sirt4, increased cellular NADPH levels and partially rescued cuproptosis. Inhibition of NAD+ kinase (NADK), which is responsible for generating NADPH, abolished the rescuing function of NMN and sirt2 for Es/Cu induced cell death. Taken together, our results suggested that DNA damage is a characteristic feature of cuproptosis. NMN can partially rescue cuproptosis by upregulating sirt2, increase intracellular NADPH content and maintain the level of Fe-S proteins, independent of the lipoamidase activity of sirt2.

Deacetylase SIRT2 Inhibition Promotes Microglial M2 Polarization Through Axl/PI3K/AKT to Alleviate White Matter Injury After Subarachnoid Hemorrhage.

White matter injury (WMI) subsequent to subarachnoid hemorrhage (SAH) frequently leads to an unfavorable patient prognosis. Previous studies have indicated that microglial M1 polarization following SAH results in the accumulation of amyloid precursor protein (APP) and degradation of myelin basic protein (MBP), thereby catalyzing the exacerbation of WMI. Consequently, transitioning microglial polarization towards the M2 phenotype (neuroprotective state) represents a potential therapeutic approach for reversing WMI. The SIRT2 gene is pivotal in neurological disorders such as neurodegeneration and ischemic stroke. However, its function and underlying mechanisms in SAH, particularly how it influences microglial function to ameliorate WMI, remain unclear. Our investigations revealed that in post-SAH, there was a temporal increase in SIRT2 expression, predominantly in the cerebral corpus callosum area, with notable colocalization with microglia. However, following the administration of the SIRT2 inhibitor AK-7, a shift in microglial polarization towards the M2 phenotype and an improvement in both short-term and long-term neuronal functions in rats were observed. Mechanistically, CO-IP experiments confirmed that SIRT2 can interact with the receptor tyrosine kinase Axl within the TAM receptor family and act as a deacetylase to regulate the deacetylation of Axl. Concurrently, the inhibition of SIRT2 by AK-7 can lead to increased expression of Axl and activation of the anti-inflammatory pathway PI3K/Akt signaling pathway, which regulates microglial M2 polarization and consequently reduces WMI. However, when Axl expression was inhibited by the injection of the shAxl virus into the lateral ventricles, the downstream signaling pathways were significantly suppressed. Rescue experiments also confirmed that the neuroprotective effects of AK-7 can be reversed by PI3K inhibitors. These data suggest that SIRT2 influences WMI by affecting microglial polarization through the Axl/PI3K/AKT pathway, and that AK-7 could serve as an effective therapeutic drug for improving neurological functions in SAH patients.

SIRT2 regulates high mobility group protein B1 nucleoplasmic shuttle and degradation via deacetylation in microglia.

High mobility group protein B1 (HMGB1) acts as a pathogenic inflammatory response to mediate ranges of conditions such as epilepsy, septic shock, ischemia, traumatic brain injury, Parkinson's disease, Alzheimer's disease and mass spectrometry. HMGB1 promotes inflammation during sterile and infectious damage and plays a crucial role in disease development. Mobilization from the nucleus to the cytoplasm is the first important step in the release of HMGB1 from activated immune cells. Here, we demonstrated that Sirtuin 2 (SIRT2) physically interacts with and deacetylates HMGB1 at 43 lysine residue at nuclear localization signal locations, strengthening its interaction with HMGB1 and causing HMGB1 to be localized in the cytoplasm. These discoveries are the first to shed light on the SIRT2 nucleoplasmic shuttle, which influences HMGB1 and its degradation, hence revealing novel therapeutic targets and avenues for neuroinflammation treatment.

FOXK2 facilitates the airway remodeling during chronic asthma by promoting glycolysis in a SIRT2-dependent manner.

Asthma is a chronic pulmonary disease with the worldwide prevalence. The structural alterations of airway walls, termed as "airway remodeling", are documented as the core contributor to the airway dysfunction during chronic asthma. Forkhead box transcription factor FOXK2 is a critical regulator of glycolysis, a metabolic reprogramming pathway linked to pulmonary fibrosis. However, the role of FOXK2 in asthma waits further explored. In this study, the chronic asthmatic mice were induced via ovalbumin (OVA) sensitization and repetitive OVA challenge. FOXK2 was upregulated in the lungs of OVA mice and downregulated after adenovirus-mediated FOXK2 silencing. The lung inflammation, peribronchial collagen deposition, and glycolysis in OVA mice were obviously attenuated after FOXK2 knockdown. Besides, the expressions of FOXK2 and SIRT2 in human bronchial epithelial cells (BEAS-2B) were increasingly upregulated upon TGF-ß1 stimulation and downregulated after FOXK2 knockdown. Moreover, the functional loss of FOXK2 remarkably suppressed TGF-ß1-induced epithelial-mesenchymal transition (EMT) and glycolysis in BEAS-2B cells, as manifested by the altered expressions of EMT markers and glycolysis enzymes. The glycolysis inhibitor 2-deoxy-d-glucose (2-DG) inhibited the EMT in TGF-ß1-induced cells, making glycolysis a driver of EMT. The binding of FOXK2 to SIRT2 was validated, and SIRT2 overexpression blocked the FOXK2 knockdown-mediated inhibition of EMT and glycolysis in TGF-ß1-treated cells, which suggests that FOXK2 regulates EMT and glycolysis in TGF-ß1-treated cells in a SIRT2-dependnet manner. Collectively, this study highlights the protective effect of FOXK2 knockdown on airway remodeling during chronic asthma.

The role of NAD-dependent deacetylase sirtuin-2 in liver metabolic stress through regulating pyruvate kinase M2 ubiquitination.

NAD-dependent deacetylase Sirt2 is involved in mammalian metabolic activities, matching energy demand with energy production and expenditure, and is relevant to a variety of metabolic diseases. Here, we constructed Sirt2 knockout and adeno-associated virus overexpression mice and found that deletion of hepatic Sirt2 accelerated primary obesity and insulin resistance in mice with concomitant hepatic metabolic dysfunction. However, the key targets of Sirt2 are unknown. We identified the M2 isoform of pyruvate kinase (PKM2) as a key Sirt2 target involved in glycolysis in metabolic stress. Through yeast two-hybrid and mass spectrometry combined with multi-omics analysis, we identified candidate acetylation modification targets of Sirt2 on PKM2 lysine 135 (K135). The Sirt2-mediated deacetylation-ubiquitination switch of PKM2 regulated the development of glycolysis. Here, we found that Sirt2 deficiency led to impaired glucose tolerance and insulin resistance and induced primary obesity. Sirt2 severely disrupted liver function in mice under metabolic stress, exacerbated the metabolic burden on the liver, and affected glucose metabolism. Sirt2 underwent acetylation modification of lysine 135 of PKM2 through a histidine 187 enzyme active site-dependent effect and reduced ubiquitination of the K48 ubiquitin chain of PKM2. Our findings reveal that the hepatic glucose metabolism links nutrient state to whole-body energetics through the rhythmic regulation of Sirt2.

1,8-cineole ameliorates experimental diabetic angiopathy by inhibiting NLRP3 inflammasome-mediated pyroptosis in HUVECs via SIRT2.

Accumulating evidence strongly support the key role of NLRP3-mediated pyroptosis in the pathogenesis and progression of vascular endothelial dysfunction associated with diabetes mellitus. Various studies have demonstrated that the activation or upregulation of Silent Information Regulation 2 homolog 2 (SIRT2) exerts inhibitory effect on the expression of NLRP3. Although 1,8-cineole has been found to protect against endothelial dysfunction and cardiovascular diseases, its role and mechanism in diabetic angiopathy remain unknown. Therefore, the aim of this study was to investigate the ameliorative effect of 1,8-cineole through SIRT2 on pyroptosis associated with diabetic angiopathy in human umbilical vein endothelial cells (HUVECs) and to elucidate the underlying mechanism. The findings revealed that 1,8-cineole exhibited a protective effect against vascular injury and ameliorated pathological alterations in the thoracic aorta of diabetic mice. Moreover, it effectively mitigated pyroptosis induced by palmitic acid-high glucose (PA-HG) in HUVECs. Treatment with 1,8-cineole effectively restored the reduced levels of SIRT2 and suppressed the elevated expression of pyroptosis-associated proteins. Additionally, our findings demonstrated the occurrence of NLRP3 deacetylation and the physical interaction between NLRP3 and SIRT2. The SIRT2 inhibitor AGK2 and siRNA-SIRT2 effectively attenuated the effect of 1,8-cineole on NLRP3 deacetylation in HUVECs and compromised its inhibitory effect against pyroptosis in HUVECs. However, overexpression of SIRT2 inhibited PA-HG-induced pyroptosis in HUVECs. 1,8-Cineole inhibited the deacetylation of NLRP3 by regulating SIRT2, thereby reducing pyroptosis in HUVECs. In conclusion, our findings suggest that PA-HG-induced pyroptosis in HUVECs plays a crucial role in the development of diabetic angiopathy, which can be mitigated by 1,8-cineole.

SIRT2 as a Potential Biomarker in Lung Adenocarcinoma: Implications for Immune Infiltration.

SIRT2 play important roles in cell cycle and cellular metabolism in the development of non-small cell lung cancer (NSCLC), and SIRT2 exhibits its therapeutic effect on NSCLC tumors with high expression of SIRT2. Nevertheless, the clinical relevance of SIRT2 in lung adenocarcinoma (LUAD), particularly its impact on tumor growth and prognostic implications, remains obscure. This investigation entailed a comprehensive analysis of SIRT2 mRNA and protein expression levels in diverse tumor and corresponding healthy tissues, utilizing databases such as TIMER 2.0, UALCAN, and HPA. Prognostic correlations of SIRT2 expression in LUAD patients, stratified by distinct clinicopathological characteristics, were evaluated using the KM Plotter database. Additionally, the TCGA and TIMER 2.0 databases were employed to assess the relationship between SIRT2 and immune infiltration, as well as to calculate immunity, stromal, and estimation scores, thus elucidating the role of SIRT2 in modulating tumor immunotherapy responses. Furthermore, Gene Set Enrichment Analysis (GSEA) was utilized to elucidate SIRT2's biological functions in pan-cancer cells. Our findings revealed a marked reduction in both mRNA and protein levels of SIRT2 in LUAD tumors relative to healthy tissue. Survival analysis indicated that diminished SIRT2 expression correlates with adverse prognostic outcomes in LUAD. Furthermore, SIRT2 expression demonstrated a significant association with various clinicopathologic attributes of LUAD patients, influencing survival outcomes across different clinicopathologic states. Functional enrichment analyses highlighted SIRT2's involvement in cell cycle regulation and immune response. Notably, SIRT2 exhibited a positive correlation with immune cell infiltration, including natural killer (NK) cells, macrophages, and dendritic cells (DCs). In summary, SIRT2 was a potential prognostic biomarker for LUAD and and a new immunotherapy target.

SIRT2-dependent DKK1 deacetylation aggravates polycystic ovary syndrome by targeting the TGF-ß1/Smad3 signaling pathway.

BACKGROUND: Polycystic ovarian syndrome (PCOS) is a prevalent metabolic and endocrine condition in females of reproductive age. This work was to discover the underlying role of Dickkopf 1 (DKK1) and its putative regulating mechanism in P COS. METHODS: Mice recieved dehydroepiandrosterone (DHEA) injection to establish the in vivo P COS model.Hematoxylin and eosin (H&E) staining was performed for histological analysis. RT-qP CR and Western blotting were used to detect gene and protein expression. CCK-8 and flow cytometry assays were applied to detect cell viability and apoptosis. Co-immunoprecipitation (Co-IP) and immunoprecipitation (IP) were applied to assess association between DKK1 and SIRT2. RESULTS: In this work, DKK1 is downregulated in P COS rats. It was revealed that DKK1 knockdown induced apoptosis and suppressed proliferation in KGN cells, whereas DKK1 overexpression had exactly the opposite effects. In addition, DKK1 deactivates the T GF-ß1/SMad3 signaling pathway, thereby controlling KGN cell proliferation and apoptosis. Besides, SIRT2 inhibition reversed the impact of DKK1 overexpression on KGN cell proliferation and apoptosis. Furthermore, SIRT2 downregulated DKK1 expression by deacetylating DKK1 in KGN cells. DISCUSSION: Altogether, we concluded that SIRT2-induced deacetylation of DKK1 triggers T GF-ß1/Smad3 hyperactivation, thereby inhibiting proliferation and promoting apoptosis of KGN cells. The above results indicated that DKK1 might function as a latent target for P COS treatment.

Recent Advances in the Discovery of SIRT1/2 Inhibitors via Computational Methods: A Perspective.

Sirtuins (SIRTs) are classified as class III histone deacetylases (HDACs), a family of enzymes that catalyze the removal of acetyl groups from the ε-N-acetyl lysine residues of histone proteins, thus counteracting the activity performed by histone acetyltransferares (HATs). Based on their involvement in different biological pathways, ranging from transcription to metabolism and genome stability, SIRT dysregulation was investigated in many diseases, such as cancer, neurodegenerative disorders, diabetes, and cardiovascular and autoimmune diseases. The elucidation of a consistent number of SIRT-ligand complexes helped to steer the identification of novel and more selective modulators. Due to the high diversity and quantity of the structural data thus far available, we reviewed some of the different ligands and structure-based methods that have recently been used to identify new promising SIRT1/2 modulators. The present review is structured into two sections: the first includes a comprehensive perspective of the successful computational approaches related to the discovery of SIRT1/2 inhibitors (SIRTIs); the second section deals with the most interesting SIRTIs that have recently appeared in the literature (from 2017). The data reported here are collected from different databases (SciFinder, Web of Science, Scopus, Google Scholar, and PubMed) using "SIRT", "sirtuin", and "sirtuin inhibitors" as keywords.

Screening approach by a combination of computational and in vitro experiments: identification of fluvastatin sodium as a potential SIRT2 inhibitor.

BACKGROUND: Sirtuins (SIRTs) are NAD+-dependent deacetylases that play various roles in numerous pathophysiological processes, holding promise as therapeutic targets worthy of further investigation. Among them, the SIRT2 subtype is closely associated with tumorigenesis and malignancies. Dysregulation of SIRT2 activation can regulate the expression levels of related genes in cancer cells, leading to tumor occurrence and metastasis. METHODS: In this study, we used computer simulations to screen for novel SIRT2 inhibitors from the FDA database, based on which 10 compounds with high docking scores and good interactions were selected for in vitro anti-pancreatic cancer metastasis testing and enzyme binding inhibition experiments. The results showed that fluvastatin sodium may possess inhibitory activity against SIRT2. Subsequently, fluvastatin sodium was subjected to molecular docking experiments with various SIRT isoforms, and the combined results from Western blotting experiments indicated its potential as a SIRT2 inhibitor. Next, molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations were performed, revealing the binding mode of fluvastatin sodium at the SIRT2 active site, further validating the stability and interaction of the ligand-protein complex under physiological conditions. RESULTS: Overall, this study provides a systematic virtual screening workflow for the discovery of SIRT2 activity inhibitors, identifies the potential inhibitory effect of fluvastatin sodium as a lead compound on SIRT2, and opens up a new direction for developing highly active and selectively targeted SIRT2 inhibitors.

An allosteric inhibitor of sirtuin 2 blocks hepatitis B virus covalently closed circular DNA establishment and its transcriptional activity.

296 million people worldwide are predisposed to developing severe end-stage liver diseases due to chronic hepatitis B virus (HBV) infection. HBV forms covalently closed circular DNA (cccDNA) molecules that persist as episomal DNA in the nucleus of infected hepatocytes and drive viral replication. Occasionally, the HBV genome becomes integrated into host chromosomal DNA, a process that is believed to significantly contribute to circulating HBsAg levels and HCC development. Neither cccDNA accumulation nor expression from integrated HBV DNA are directly targeted by current antiviral treatments. In this study, we investigated the antiviral properties of a newly described allosteric modulator, FLS-359, that targets sirtuin 2 (SIRT2), an NAD+-dependent deacylase. Our results demonstrate that SIRT2 modulation by FLS-359 and by other tool compounds inhibits cccDNA synthesis following de novo infection of primary human hepatocytes and HepG2 (C3A)-NTCP cells, and FLS-359 substantially reduces cccDNA recycling in HepAD38 cells. While pre-existing cccDNA is not eradicated by short-term treatment with FLS-359, its transcriptional activity is substantially impaired, likely through inhibition of viral promoter activities. Consistent with the inhibition of viral transcription, HBsAg production by HepG2.2.15 cells, which contain integrated HBV genomes, is also suppressed by FLS-359. Our study provides further insights on SIRT2 regulation of HBV infection and supports the development of potent SIRT2 inhibitors as HBV antivirals.

New insight into arginine and tryptophan metabolism in macrophage activation during tuberculosis.

Arginine and tryptophan are pivotal in orchestrating cytokine-driven macrophage polarization and immune activation. Specifically, interferon-gamma (IFN-γ) stimulates inducible nitric oxide synthase (iNOS) expression), leading to the conversion of arginine into citrulline and nitric oxide (NO), while Interleukin-4 (IL4) promotes arginase activation, shifting arginine metabolism toward ornithine. Concomitantly, IFN-γ triggers indoleamine 2,3-dioxygenase 1 (IDO1) and Interleukin-4 induced 1 (IL4i1), resulting in the conversion of tryptophan into kynurenine and indole-3-pyruvic acid. These metabolic pathways are tightly regulated by NAD+-dependent sirtuin proteins, with Sirt2 and Sirt5 playing integral roles. In this review, we present novel insights that augment our understanding of the metabolic pathways of arginine and tryptophan following Mycobacterium tuberculosis infection, particularly their relevance in macrophage responses. Additionally, we discuss arginine methylation and demethylation and the role of Sirt2 and Sirt5 in regulating tryptophan metabolism and arginine metabolism, potentially driving macrophage polarization.

NAD+ precursors promote the restoration of spermatogenesis in busulfan-treated mice through inhibiting Sirt2-regulated ferroptosis.

Rationale: In recent years, nicotinamide adenine dinucleotide (NAD+) precursors (Npre) have been widely employed to ameliorate female reproductive problems in both humans and animal models. However, whether and how Npre plays a role in the male reproductive disorder has not been fully clarified. Methods: In the present study, a busulfan-induced non-obstructive azoospermic mouse model was used, and Npre was administered for five weeks following the drug injection, with the objective of reinstating spermatogenesis and fertility. Initially, we assessed the NAD+ level, germ cell types, semen parameters and sperm fertilization capability. Subsequently, testis tissues were examined through RNA sequencing analysis, ELISA, H&E, immunofluorescence, quantitative real-time PCR, and Western blotting techniques. Results: The results indicated that Npre restored normal level of NAD+ in blood and significantly alleviated the deleterious effects of busulfan (BU) on spermatogenesis, thereby partially reestablishing fertilization capacity. Transcriptome analysis, along with recovery of testicular Fe2+, GSH, NADPH, and MDA levels, impaired by BU, and the fact that Fer-1, an inhibitor of ferroptosis, restored spermatogenesis and semen parameters close to CTRL values, supported such possibility. Interestingly, the reduction in SIRT2 protein level by the specific inhibitor AGK2 attenuated the beneficial effects of Npre on spermatogenesis and ferroptosis by affecting PGC-1α and ACLY protein levels, thus suggesting how these compounds might confer spermatogenesis protection. Conclusion: Collectively, these findings indicate that NAD+ protects spermatogenesis against ferroptosis, probably through SIRT2 dependent mechanisms. This underscores the considerable potential of Npre supplementation as a feasible strategy for preserving or restoring spermatogenesis in specific conditions of male infertility and as adjuvant therapy to preserve male fertility in cancer patients receiving sterilizing treatments.

Ligand-based design and synthesis of new trityl histamine and trityl cysteamine derivatives as SIRT2 inhibitors for cancer therapy.

The relentless pursuit of novel therapeutic agents against cancer has led to the identification of multiple molecular targets, among which Sirtuin 2 (SIRT2) has garnered significant attention. This study presents an extensive SAR study of our reported trityl scaffold-based SIRT2 inhibitors. This study encompasses a range of different medicinal chemistry approaches to improve the activity of the lead compounds TH-3 and STCY1. The rationally designed and synthesized structures were confirmed using NMR and high-resolution mass spectroscopy before performing SIRT2 inhibition assay, NCI60 cytotoxicity test, and cell cycle analysis. Indeed, our strategies afforded hitherto unreported SIRT2 inhibitors with high activity, particularly 2a, 4a, 7c, and 7f. Remarkably, the presence of a lipophilic para substitution on the phenyl group of a freely rotating or a locked trityl moiety enhanced activity SIRT2 inhibition. Concomitantly, the synthesized compounds showed prominent activity against different cancer lines from the NCI60 assay. Of interest, compound 7c stands out as a potent and highly selective antiproliferative agent against leukemia and colon cancer panels. Furthermore, 7c treatment resulted in cell cycle arrest in MCF-7 cells at G2 phase and did not cause in vitro DNA cleavage.

m6A-Induced lncRNA MEG3 Promotes Cerebral Ischemia-Reperfusion Injury Via Modulating Oxidative Stress and Mitochondrial Dysfunction by hnRNPA1/Sirt2 Axis.

Ischemic stroke remains one of the major causes of serious disability and death globally. LncRNA maternally expressed gene 3 (MEG3) is elevated in middle cerebral artery occlusion/reperfusion (MCAO/R) rats and oxygen-glucose deprivation/reperfusion (OGD/R)-treated neurocytes cells. The objective of this study is to investigate the mechanism underlying MEG3-regulated cerebral ischemia/reperfusion (I/R) injury. MCAO/R mouse model and OGD/R-treated HT-22 cell model were established. The cerebral I/R injury was monitored by TTC staining, neurological scoring, H&E and TUNEL assay. The levels of MEG3, hnRNPA1, Sirt2 and other key molecules were detected by qRT-PCR and western blot. Mitochondrial dysfunction was assessed by transmission Electron Microscopy (TEM), JC-1 and MitoTracker staining. Oxidative stress was monitored using commercial kits. Bioinformatics analysis, RIP, RNA pull-down assays and RNA FISH were employed to detect the interactions among MEG3, hnRNPA1 and Sirt2. The m6A modification of MEG3 was assessed by MeRIP-qPCR. MEG3 promoted MCAO/R-induced brain injury by modulating mitochondrial fragmentation and oxidative stress. It also facilitated OGD/R-induced apoptosis, mitochondrial dysfunction and oxidative stress in HT-22 cells. Mechanistically, direct associations between MEG3 and hnRNPA1, as well as between hnRNPA1 and Sirt2, were observed in HT-22 cells. MEG3 regulated Sirt2 expression in a hnRNPA1-dependent manner. Functional studies showed that MEG3/Sirt2 axis contributed to OGD/R-induced mitochondrial dysfunction and oxidative stress in HT-22 cells. Additionally, METTL3 was identified as the m6A transferase responsible for the m6A modification of MEG3. m6A-induced lncRNA MEG3 promoted cerebral I/R injury via modulating oxidative stress and mitochondrial dysfunction by hnRNPA1/Sirt2 axis.