Utilizing machine learning to identify nifuroxazide as an inhibitor of ubiquitin-specific protease 21 in a drug repositioning strategy.

Ubiquitin-specific protease (USP), an enzyme catalyzing protein deubiquitination, is involved in biological processes related to metabolic disorders and cancer proliferation. We focused on constructing predictive models tailored to unveil compounds boasting USP21 inhibitory attributes. Six models, Extra Trees Classifier, Random Forest Classifier, LightGBM Classifier, XGBoost Classifier, Bagging Classifier, and a convolutional neural network harnessed from empirical data were selected for the screening process. These models guided our selection of 26 compounds from the FDA-approved drug library for further evaluation. Notably, nifuroxazide emerged as the most potent inhibitor, with a half-maximal inhibitory concentration of 14.9 ± 1.63 µM. The stability of protein-ligand complexes was confirmed using molecular modeling. Furthermore, nifuroxazide treatment of HepG2 cells not only inhibited USP21 and its established substrate ACLY but also elevated p-AMPKα, a downstream functional target of USP21. Intriguingly, we unveiled the previously unknown capacity of nifuroxazide to increase the levels of miR-4458, which was identified as downregulating USP21. This discovery was substantiated by manipulating miR-4458 levels in HepG2 cells, resulting in corresponding changes in USP21 protein levels in line with its predicted interaction with ACLY. Lastly, we confirmed the in vivo efficacy of nifuroxazide in inhibiting USP21 in mice livers, observing concurrent alterations in ACLY and p-AMPKα levels. Collectively, our study establishes nifuroxazide as a promising USP21 inhibitor with potential implications for addressing metabolic disorders and cancer proliferation. This multidimensional investigation sheds light on the intricate regulatory mechanisms involving USP21 and its downstream effects, paving the way for further exploration and therapeutic development.

Identification of lignans as ATP citrate lyase (ACLY) inhibitors from the roots of Pouzolzia zeylanica.

A new lignan, named pouzolignan P (1), together with 14 known ones (2 - 15) were isolated from the roots of Pouzolzia zeylanica (L.) Benn. Their structures were deduced based on the detailed spectroscopic analysis. All the isolates were evaluated for their inhibitory activities toward the ATP citrate lyase (ACLY). Among them, four lignans, isopouzolignan K (3), gnemontanins E (5), gnetuhainin I (6), and styraxlignolide D (15) showed excellent ACLY inhibitory effect with IC50 values of 9.06, 0.59, 2.63, and 7.62 µM, respectively. These compounds were further evaluated for their cholesterol-lowing effects on ox-LDL-induced high-cholesterol HepG2 cells. Compound 15 emerges as the most potent ACLY inhibitor, which significantly decreased the TC level in a dose-dependent manner. In addition, molecular docking simulations elucidated that 15 formed a strong hydrogen-bond interaction with Glu599 of ACLY, which was an important site responsible for the enzyme catalytic activity.

Combined Inhibition of UBE2C and PLK1 Reduce Cell Proliferation and Arrest Cell Cycle by Affecting ACLY in Pan-Cancer.

Various studies have shown that the cell-cycle-related regulatory proteins UBE2C, PLK1, and BIRC5 promote cell proliferation and migration in different types of cancer. However, there is a lack of in-depth and systematic research on the mechanism of these three as therapeutic targets. In this study, we found a positive correlation between the expression of UBE2C and PLK1/BIRC5 in the Cancer Genome Atlas (TCGA) database, revealing a potential combination therapy candidate for pan-cancer. Quantitative real-time PCR (qRT-PCR), Western blotting (WB), cell phenotype detection, and RNA-seq techniques were used to evidence the effectiveness of the combination candidate. We found that combined interference of UBE2C with PLK1 and UBE2C with BIRC5 affected metabolic pathways by significantly downregulating the mRNA expression of IDH1 and ACLY, which was related to the synthesis of acetyl-CoA. By combining the PLK1 inhibitor volasertib and the ACLY inhibitor bempedoic acid, it showed a higher synergistic inhibition of cell viability and higher synergy scores in seven cell lines, compared with those of other combination treatments. Our study reveals the potential mechanisms through which cell-cycle-related genes regulate metabolism and proposes a potential combined targeted therapy for patients with higher PLK1 and ACLY expression in pan-cancer.

ACLY as a modulator of liver cell functions and its role in Metabolic Dysfunction-Associated Steatohepatitis.

BACKGROUND: Non-alcoholic Fatty Liver Disease (NAFLD), now better known as Metabolic (Dysfunction)-Associated Fatty Liver Disease (MAFLD) and its progression to Nonalcoholic Steatohepatitis (NASH), more recently referred to as Metabolic (Dysfunction)-Associated Steatohepatitis (MASH) are the most common causes of liver failure and chronic liver damage. The new names emphasize the metabolic involvement both in relation to liver function and pathological features with extrahepatic manifestations. This study aims to explore the role of the immunometabolic enzyme ATP citrate lyase (ACLY), with a critical function in lipogenesis, carbohydrate metabolism, gene expression and inflammation. METHODS: ACLY function was investigated in TNFα-triggered human hepatocytes and in PBMC-derived macrophages from MASH patients. Evaluation of expression levels was carried out by western blotting and/or RT-qPCR. In the presence or absence of ACLY inhibitors, ROS, lipid peroxidation and GSSG oxidative stress biomarkers were quantified. Chromatin immunoprecipitation (ChIP), transient transfections, immunocytochemistry, histone acetylation quantitation were used to investigate ACLY function in gene expression reprogramming. IL-6 and IL-1ß were quantified by Lumit immunoassays. RESULTS: Mechanistically, ACLY inhibition reverted lipid accumulation and oxidative damage while reduced secretion of inflammatory cytokines in TNFα-triggered human hepatocytes. These effects impacted not only on lipid metabolism but also on other crucial features of liver function such as redox status and production of inflammatory mediators. Moreover, ACLY mRNA levels together with those of malic enzyme 1 (ME1) increased in human PBMC-derived macrophages from MASH patients when compared to age-matched healthy controls. Remarkably, a combination of hydroxycitrate (HCA), the natural ACLY inhibitor, with red wine powder (RWP) significantly lowered ACLY and ME1 mRNA amount as well as IL-6 and IL-1ß production in macrophages from subjects with MASH. CONCLUSION: Collectively, our findings for the first time highlight a broad spectrum of ACLY functions in liver as well as in the pathogenesis of MASH and its diagnostic and therapeutic potential value.

Identification of glutamyl-prolyl-tRNA synthetase as a new therapeutic target in hepatocellular carcinoma via a novel bioinformatic approach.

Background: Hepatocellular carcinoma (HCC) has a high incidence, and current treatments are ineffective. We aimed to explore potential diagnostic and prognostic biomarkers for HCC by conducting bioinformatics analysis on genomic and proteomic data. Methods: Genome and proteome data were downloaded from The Cancer Genome Atlas (TCGA) and ProteomeXchange databases, respectively. Differentially expressed genes was determined using limma package. Functional enrichment analysis was conducted by Database for Annotation, Visualization, and Integrated Discovery (DAVID). Protein-protein analysis was established by STRING dataset. Using Cytoscope for network visualization and CytoHubba for hub gene identification. The gene mRNA and protein levels were validated using GEPIA and HPA, as well as RT-qPCR and Western blot. Results: A total of 127 up-regulated and 80 down-regulated common DEGPs were identified between the genomic and proteomic data, Mining 10 key genes/proteins(ACLY, ACACB, EPRS, CAD, HSPA4, ACACA, MTHFD1, DMGDH, ALDH2, and GLDC) through protein interaction networks. in addition, Glutamyl-prolyl-tRNA synthetase (EPRS) was highlighted as an HCC biomarker that is negatively correlated with survival. Differential EPRS expression analysis in HCC and paracancerous tissues showed that EPRS expression was elevated in HCC. RT-qPCR and Western blot analysis results showed that EPRS expression was upregulated in HCC cells. Conclusions: Our results suggest that EPRS is a potential therapeutic target for inhibiting HCC tumorigenesis and progression.

Development of the novel ACLY inhibitor 326E as a promising treatment for hypercholesterolemia.

Hepatic cholesterol accumulation is an important contributor to hypercholesterolemia, which results in atherosclerosis and cardiovascular disease (CVD). ATP-citrate lyase (ACLY) is a key lipogenic enzyme that converts cytosolic citrate derived from tricarboxylic acid cycle (TCA cycle) to acetyl-CoA in the cytoplasm. Therefore, ACLY represents a link between mitochondria oxidative phosphorylation and cytosolic de novo lipogenesis. In this study, we developed the small molecule 326E with an enedioic acid structural moiety as a novel ACLY inhibitor, and its CoA-conjugated form 326E-CoA inhibited ACLY activity with an IC50 = 5.31 ± 1.2 µmol/L in vitro. 326E treatment reduced de novo lipogenesis, and increased cholesterol efflux in vitro and in vivo. 326E was rapidly absorbed after oral administration, exhibited a higher blood exposure than that of the approved ACLY inhibitor bempedoic acid (BA) used for hypercholesterolemia. Chronic 326E treatment in hamsters and rhesus monkeys resulted in remarkable improvement of hyperlipidemia. Once daily oral administration of 326E for 24 weeks prevented the occurrence of atherosclerosis in ApoE-/- mice to a greater extent than that of BA treatment. Taken together, our data suggest that inhibition of ACLY by 326E represents a promising strategy for the treatment of hypercholesterolemia.

Development of the novel ACLY inhibitor 326E as a promising treatment for hypercholesterolemia

Hepatic cholesterol accumulation is an important contributor to hypercholesterolemia, which results in atherosclerosis and cardiovascular disease (CVD). ATP-citrate lyase (ACLY) is a key lipogenic enzyme that converts cytosolic citrate derived from tricarboxylic acid cycle (TCA cycle) to acetyl-CoA in the cytoplasm. Therefore, ACLY represents a link between mitochondria oxidative phosphorylation and cytosolic de novo lipogenesis. In this study, we developed the small molecule 326E with an enedioic acid structural moiety as a novel ACLY inhibitor, and its CoA-conjugated form 326E-CoA inhibited ACLY activity with an IC50 = 5.31 ± 1.2 μmol/L in vitro. 326E treatment reduced de novo lipogenesis, and increased cholesterol efflux in vitro and in vivo. 326E was rapidly absorbed after oral administration, exhibited a higher blood exposure than that of the approved ACLY inhibitor bempedoic acid (BA) used for hypercholesterolemia. Chronic 326E treatment in hamsters and rhesus monkeys resulted in remarkable improvement of hyperlipidemia. Once daily oral administration of 326E for 24 weeks prevented the occurrence of atherosclerosis in ApoE-/- mice to a greater extent than that of BA treatment. Taken together, our data suggest that inhibition of ACLY by 326E represents a promising strategy for the treatment of hypercholesterolemia.

Phosphoproteomic Analysis of Breast Cancer-Derived Small Extracellular Vesicles Reveals Disease-Specific Phosphorylated Enzymes.

Small membrane-derived extracellular vesicles have been proposed as participating in several cancer diseases, including breast cancer (BC). We performed a phosphoproteomic analysis of breast cancer-derived small extracellular vesicles (sEVs) to provide insight into the molecular and cellular regulatory mechanisms important for breast cancer tumor progression and metastasis. We examined three cell line models for breast cancer: MCF10A (non-malignant), MCF7 (estrogen and progesterone receptor-positive, metastatic), and MDA-MB-231 (triple-negative, highly metastatic). To obtain a comprehensive overview of the sEV phosphoproteome derived from each cell line, effective phosphopeptide enrichment techniques IMAC and TiO2, followed by LC-MS/MS, were performed. The phosphoproteome was profiled to a depth of 2003 phosphopeptides, of which 207, 854, and 1335 were identified in MCF10A, MCF7, and MDA-MB-231 cell lines, respectively. Furthermore, 2450 phosphorylation sites were mapped to 855 distinct proteins, covering a wide range of functions. The identified proteins are associated with several diseases, mostly related to cancer. Among the phosphoproteins, we validated four enzymes associated with cancer and present only in sEVs isolated from MCF7 and MDA-MB-231 cell lines: ATP citrate lyase (ACLY), phosphofructokinase-M (PFKM), sirtuin-1 (SIRT1), and sirtuin-6 (SIRT6). With the exception of PFKM, the specific activity of these enzymes was significantly higher in MDA-MB-231 when compared with MCF10A-derived sEVs. This study demonstrates that sEVs contain functional metabolic enzymes that could be further explored for their potential use in early BC diagnostic and therapeutic applications.

Bempedoic acid: effect of ATP-citrate lyase inhibition on low-density lipoprotein cholesterol and other lipids.

Bempedoic acid is a new, first-in-class oral ATP-citrate lyase (ACLY) inhibitor that has to be converted to its CoA thioester before it inhibits ACLY. This conversion only occurs in the liver and not in skeletal muscle. This may explain why, unlike the statins, bempedoic acid does not cause myalgia. Bempedoic acid given at a dosage of 180 mg orally once daily produces a highly significant reduction in low-density lipoprotein cholesterol (LDL-C), non-high-density lipoprotein cholesterol, total cholesterol, apolipoprotein B and importantly also in high-sensitivity C-reactive protein. It has recently been approved by both the Food and Drug Administration (FDA) and the European Commission for use in adult patients with heterozygous familial hypercholesterolemia or atherosclerotic cardiovascular disease who require additional lowering of LDL-C, and for the treatment of adults with primary hypercholesterolemia (heterozygous familial and nonfamilial) or mixed dyslipidemia, respectively.

FABP7 Regulates Acetyl-CoA Metabolism Through the Interaction with ACLY in the Nucleus of Astrocytes.

Fatty acid binding protein 7 (FABP7) is an intracellular fatty acid chaperon that is highly expressed in astrocytes, oligodendrocyte-precursor cells, and malignant glioma. Previously, we reported that FABP7 regulates the response to extracellular stimuli by controlling the expression of caveolin-1, an important component of lipid raft. Here, we explored the detailed mechanisms underlying FABP7 regulation of caveolin-1 expression using primary cultured FABP7-KO astrocytes as a model of loss of function and NIH-3T3 cells as a model of gain of function. We discovered that FABP7 interacts with ATP-citrate lyase (ACLY) and is important for acetyl-CoA metabolism in the nucleus. This interaction leads to epigenetic regulation of several genes, including caveolin-1. Our novel findings suggest that FABP7-ACLY modulation of nuclear acetyl-CoA has more influence on histone acetylation than cytoplasmic acetyl-CoA. The changes to histone structure may modify caveolae-related cell activity in astrocytes and tumors, including malignant glioma.

ATP-citrate lyase (ACLY) in lipid metabolism and atherosclerosis: An updated review.

ATP citrate lyase (ACLY) is an important enzyme linking carbohydrate to lipid metabolism by generating acetyl-CoA from citrate for fatty acid and cholesterol biosynthesis. Mendelian randomization of large human cohorts has validated ACLY as a promising target for low-density-lipoprotein-cholesterol (LDL-C) lowering and cardiovascular protection. Among current ACLY inhibitors, Bempedoic acid (ETC-1002) is a first-in-class, prodrug-based direct competitive inhibitor of ACLY which regulates lipid metabolism by upregulating hepatic LDL receptor (LDLr) expression and activity. ACLY deficiency in hepatocytes protects from hepatic steatosis and dyslipidemia. In addition, pharmacological inhibition of ACLY by bempedoic acid, prevents dyslipidemia and attenuates atherosclerosis in hypercholesterolemic ApoE-/- mice, LDLr-/- mice, and LDLr-/- miniature pigs. Convincing data from clinical trials have revealed that bempedoic acid significantly lowers LDL-C as monotherapy, combination therapy, and add-on with statin therapy in statin-intolerant patients. More recently, a phase 3 CLEAR Harmony clinical trial ("Safety and Efficacy of Bempedoic Acid to Reduce LDL Cholesterol") has shown that bempedoic acid reduces the level of LDL-C in hypercholesterolemic patients receiving guideline-recommended statin therapy with a good safety profile. Hereby, we provide a updated review of the expression, regulation, genetics, functions of ACLY in lipid metabolism and atherosclerosis, and highlight the therapeutic potential of ACLY inhibitors (such as bempedoic acid, SB-204990, and other naturally-occuring inhibitors) to treat atherosclerotic cardiovascular diseases. It must be pointed out that long-term large-scale clinical trials in high-risk patients, are warranted to validate whether ACLY represent a promising therapeutic target for pharmaceutic intervention of dyslipidemia and atherosclerosis; and assess the safety and efficacy profile of ACLY inhibitors in improving cardiovascular outcome of patients.

Enhanced acetylation of ATP-citrate lyase promotes the progression of nonalcoholic fatty liver disease.

Hepatic steatosis is a hallmark of nonalcoholic fatty liver disease (NAFLD) and is promoted by dysregulated de novo lipogenesis. ATP-citrate lyase (ACLY) is a crucial lipogenic enzyme that is up-regulated in individuals with NAFLD. A previous study has shown that acetylation of ACLY at Lys-540, Lys-546, and Lys-554 (ACLY-3K) increases ACLY protein stability by antagonizing its ubiquitylation, thereby promoting lipid synthesis and cell proliferation in lung cancer cells. But the functional importance of this regulatory mechanism in other cellular or tissue contexts or under other pathophysiological conditions awaits further investigation. Here, we show that ACLY-3K acetylation also promotes ACLY protein stability in AML12 cells, a mouse hepatocyte cell line, and found that the deacetylase sirtuin 2 (SIRT2) deacetylates ACLY-3K and destabilizes ACLY in these cells. Of note, the livers of mice and humans with NAFLD had increased ACLY protein and ACLY-3K acetylation levels and decreased SIRT2 protein levels. Mimicking ACLY-3K acetylation by replacing the three lysines with three glutamines (ACLY-3KQ variant) promoted lipid accumulation both in high glucose-treated AML12 cells and in the livers of high-fat/high-sucrose (HF/HS) diet-fed mice. Moreover, overexpressing SIRT2 in AML12 cells inhibited lipid accumulation, which was more efficiently reversed by overexpressing the ACLY-3KQ variant than by overexpressing WT ACLY. Additionally, hepatic SIRT2 overexpression decreased ACLY-3K acetylation and its protein level and alleviated hepatic steatosis in HF/HS diet-fed mice. Our findings reveal a posttranscriptional mechanism underlying the up-regulation of hepatic ACLY in NAFLD and suggest that the SIRT2/ACLY axis is involved in NAFLD progression.

Silencing of solute carrier family 13 member 5 disrupts energy homeostasis and inhibits proliferation of human hepatocarcinoma cells.

The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays a key role in importing citrate from the circulation into liver cells. Recent evidence has revealed that SLC13A5 deletion protects mice from high-fat diet-induced hepatic steatosis and that mutation of the SLC13A5 orthologues in Drosophila melanogaster and Caenorhabditis elegans promotes longevity. However, despite the emerging importance of SLC13A5 in energy homeostasis, whether perturbation of SLC13A5 affects the metabolism and malignancy of hepatocellular carcinoma is unknown. Here, we sought to determine whether SLC13A5 regulates hepatic energy homeostasis and proliferation of hepatoma cells. RNAi-mediated silencing of SLC13A5 expression in two human hepatoma cell lines, HepG2 and Huh7, profoundly suppressed cell proliferation and colony formation, and induced cell cycle arrest accompanied by increased expression of cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin B1. Furthermore, such suppressive effects were also observed on the growth of HepG2 cell-derived xenografts expressing SLC13A5-shRNA in nude mice. Metabolically, knockdown of SLC13A5 in HepG2 and Huh7 cells was associated with a decrease in intracellular levels of citrate, the ratio of ATP/ADP, phospholipid content, and ATP citrate lyase expression. Moreover, both in vitro and in vivo assays demonstrated that SLC13A5 depletion promotes activation of the AMP-activated protein kinase, which was accompanied by deactivation of oncogenic mechanistic target of rapamycin signaling. Together, our findings expand the role of SLC13A5 from facilitating hepatic energy homeostasis to influencing hepatoma cell proliferation and suggest a potential role of SLC13A5 in the progression of human hepatocellular carcinoma.

ATP-citrate lyase regulates cellular senescence via an AMPK- and p53-dependent pathway.

ATP citrate lyase (ACLY) is a key enzyme that is involved in de novo lipogenesis by catalyzing conversion of cytosolic citrate into acetyl CoA and oxaloacetate. Up-regulation of ACLY in various types of tumors enhances fatty acid synthesis and supplies excess acetyl CoA for histone acetylation. However, there is evidence that its enzymatic activity alone is insufficient to explain ACLY silencing-mediated growth arrest in tumor cells. In this study, we found that ACLY knockdown in primary human cells triggers cellular senescence and activation of tumor suppressor p53. Provision of acetyl CoA to ACLY knockdown cells did not alleviate ACLY silencing-induced p53 activation, suggesting an independent role for ACLY activity. Instead, ACLY physically interacted with the catalytic subunit of AMP-activated protein kinase (AMPK) and inhibited AMPK activity. The activation of AMPK under ACLY knockdown conditions may lead to p53 activation, ultimately leading to cellular senescence. In cancer cells, ACLY silencing-induced p53 activation facilitated DNA damage-induced cell death. Taken together, our results suggest a novel function of ACLY in cellular senescence and tumorigenesis.