Exploring the Role of Bempedoic Acid in Metabolic Dysfunction Associated Steatotic Liver Disease: Actual Evidence and Future Perspectives.

Metabolic dysfunction-associated steatotic liver disease (MASLD) involves excessive lipid accumulation in hepatocytes, impacting global healthcare due to its high prevalence and risk of progression to severe liver conditions. Its pathogenesis involves genetic, metabolic, and inflammatory factors, with cardiovascular events as the leading cause of mortality. This review examines the role of lipid-lowering therapies in MASLD, with a particular focus on bempedoic acid, a recently approved cholesterol-lowering agent for hypercholesterolemia and high cardiovascular-risk patients. It explores its potential in liver disease by modulating lipid metabolism and inflammatory pathways based on the most recent studies available. Bempedoic acid inhibits ATP-citrate lyase, reducing cholesterol and fatty acid synthesis while activating AMP-activated protein kinase to suppress gluconeogenesis and lipogenesis. Animal studies indicate its efficacy in reducing hepatic steatosis, inflammation, and fibrosis. Bempedoic acid holds promise as a therapeutic for MASLD, offering dual benefits in lipid metabolism and inflammation. Further clinical trials are required to confirm its efficacy and safety in MASLD patients, potentially addressing the multifaceted nature of this disease.

Citrate metabolism controls the senescent microenvironment via the remodeling of pro-inflammatory enhancers.

The senescent microenvironment and aged cells per se contribute to tissue remodeling, chronic inflammation, and age-associated dysfunction. However, the metabolic and epigenomic bases of the senescence-associated secretory phenotype (SASP) remain largely unknown. Here, we show that ATP-citrate lyase (ACLY), a key enzyme in acetyl-coenzyme A (CoA) synthesis, is essential for the pro-inflammatory SASP, independent of persistent growth arrest in senescent cells. Citrate-derived acetyl-CoA facilitates the action of SASP gene enhancers. ACLY-dependent de novo enhancers augment the recruitment of the chromatin reader BRD4, which causes SASP activation. Consistently, specific inhibitions of the ACLY-BRD4 axis suppress the STAT1-mediated interferon response, creating the pro-inflammatory microenvironment in senescent cells and tissues. Our results demonstrate that ACLY-dependent citrate metabolism represents a selective target for controlling SASP designed to promote healthy aging.

Hydroxycitrate delays early mortality in mice and promotes muscle regeneration while inducing a rich hepatic energetic status.

ATP citrate lyase (ACLY) inhibitors have the potential of modulating central processes in protein, carbohydrate, and lipid metabolism, which can have relevant physiological consequences in aging and age-related diseases. Here, we show that hepatic phospho-active ACLY correlates with overweight and Model for End-stage Liver Disease score in humans. Wild-type mice treated chronically with the ACLY inhibitor potassium hydroxycitrate exhibited delayed early mortality. In AML12 hepatocyte cultures, the ACLY inhibitors potassium hydroxycitrate, SB-204990, and bempedoic acid fostered lipid accumulation, which was also observed in the liver of healthy-fed mice treated with potassium hydroxycitrate. Analysis of soleus tissue indicated that potassium hydroxycitrate produced the modulation of wound healing processes. In vivo, potassium hydroxycitrate modulated locomotor function toward increased wire hang performance and reduced rotarod performance in healthy-fed mice, and improved locomotion in mice exposed to cardiotoxin-induced muscle atrophy. Our findings implicate ACLY and ACLY inhibitors in different aspects of aging and muscle regeneration.

CILP2 promotes hypertrophic scar through Snail acetylation by interaction with ACLY.

BACKGROUND & AIMS: Hypertrophic scar (HS) is a skin fibroproliferative disorder occurring after burns, surgeries or traumatic injuries, and it has caused a tremendous economic and medical burden. Its molecular mechanism is associated with the abnormal proliferation and transition of fibroblasts and excessive deposition of extracellular matrix. Cartilage intermediate layer protein 2 (CILP2), highly homologous to cartilage intermediate layer protein 1 (CILP1), is mainly secreted predominantly from chondrocytes in the middle/deeper layers of articular cartilage. Recent reports indicate that CILP2 is involved in the development of fibrotic diseases. We investigated the role of CILP2 in the progression of HS. METHODS AND RESULTS: It was found in this study that CILP2 expression was significantly higher in HS than in normal skin, especially in myofibroblasts. In a clinical cohort, we discovered that CILP2 was more abundant in the serum of patients with HS, especially in the early stage of HS. In vitro studies indicated that knockdown of CILP2 suppressed proliferation, migration, myofibroblast activation and collagen synthesis of hypertrophic scar fibroblasts (HSFs). Further, we revealed that CILP2 interacts with ATP citrate lyase (ACLY), in which CILP2 stabilizes the expression of ACLY by reducing the ubiquitination of ACLY, therefore prompting Snail acetylation and avoiding reduced expression of Snail. In vivo studies indicated that knockdown of CILP2 or ACLY inhibitor, SB-204990, significantly alleviated HS formation. CONCLUSION: CILP2 exerts a vital role in hypertrophic scar formation and might be a detectable biomarker reflecting the progression of hypertrophic scar and a therapeutic target for hypertrophic scar.

Discovery and characterization of novel ATP citrate lyase inhibitors from Acanthopanax senticosus (Rupr. & Maxim.) Harms.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism, and abnormally high expression of ACLY occurs in many diseases, including cancers, dyslipidemia and cardiovascular diseases. ACLY inhibitors are prospective treatments for these diseases. However, the scaffolds of ACLY inhibitors are insufficient with weak activity. The discovery of inhibitors with structural novelty and high activity continues to be a research hotpot. Acanthopanax senticosus (Rupr. & Maxim.) Harms is used for cardiovascular disease treatment, from which no ACLY inhibitors have ever been found. In this work, we discovered three novel ACLY inhibitors, and the most potent one was isochlorogenic acid C (ICC) with an IC50 value of 0.14 ± 0.04 µM. We found dicaffeoylquinic acids with ortho-dihydroxyphenyl groups were important features for inhibition by studying ten phenolic acids. We further investigated interactions between the highly active compound ICC and ACLY. Thermal shift assay revealed that ICC could directly bind to ACLY and improve its stability in the heating process. Enzymatic kinetic studies indicated ICC was a noncompetitive inhibitor of ACLY. Our work discovered novel ACLY inhibitors, provided valuable structure-activity patterns and deepened knowledge on the interactions between this targe tand its inhibitors.

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.

13C-SpaceM: Spatial single-cell isotope tracing reveals heterogeneity of de novo fatty acid synthesis in cancer.

Metabolism has emerged as a key factor in homeostasis and disease including cancer. Yet, little is known about the heterogeneity of metabolic activity of cancer cells due to the lack of tools to directly probe it. Here, we present a novel method, 13C-SpaceM for spatial single-cell isotope tracing of glucose-dependent de novo lipogenesis. The method combines imaging mass spectrometry for spatially-resolved detection of 13C6-glucose-derived 13C label incorporated into esterified fatty acids with microscopy and computational methods for data integration and analysis. We validated 13C-SpaceM on a spatially-heterogeneous normoxia-hypoxia model of liver cancer cells. Investigating cultured cells, we revealed single-cell heterogeneity of lipogenic acetyl-CoA pool labelling degree upon ACLY knockdown that is hidden in the bulk analysis and its effect on synthesis of individual fatty acids. Next, we adapted 13C-SpaceM to analyze tissue sections of mice harboring isocitrate dehydrogenase (IDH)-mutant gliomas. We found a strong induction of de novo fatty acid synthesis in the tumor tissue compared to the surrounding brain. Comparison of fatty acid isotopologue patterns revealed elevated uptake of mono-unsaturated and essential fatty acids in the tumor. Furthermore, our analysis uncovered substantial spatial heterogeneity in the labelling of the lipogenic acetyl-CoA pool indicative of metabolic reprogramming during microenvironmental adaptation. Overall, 13C-SpaceM enables novel ways for spatial probing of metabolic activity at the single cell level. Additionally, this methodology provides unprecedented insight into fatty acid uptake, synthesis and modification in normal and cancerous tissues.

Nobiletin targets SREBP1/ACLY to induce autophagy-dependent cell death of gastric cancer cells through PI3K/Akt/mTOR signaling pathway.

BACKGROUND: Autophagy could sense metabolic conditions and safeguard cells against nutrient deprivation, ultimately supporting the survival of cancer cells. Nobiletin (NOB) is a kind of bioactive component of the traditional Chinese medicine Citri Reticulatae Pericarpium and has been proven to induce GC cell death by reducing de novo fatty acid synthesis in our previous study. Nevertheless, the precise mechanisms by which NOB induces cell death in GC cells still need further elucidation. OBJECTIVES: To examine the mechanism by which NOB inhibits gastric cancer progression through the regulation of autophagy under the condition of lipid metabolism inhibition. METHODS/ STUDY DESIGN: Proliferation was detected by the CCK-8 assay. RNA sequencing (RNA-seq) was used to examine signaling pathway changes. Electron microscopy and mRFP-GFP-LC3 lentiviral transfection were performed to observe autophagy in vitro. Western blot, plasmid transfection, immunofluorescence staining, and CUT & Tag-qPCR techniques were utilized to explore the mechanisms by which NOB affects GC cells. Molecular docking and molecular dynamics simulations were conducted to predict the binding mode of NOB and SREBP1. CETSA was adopted to verify the predicted of binding model. A patient-derived xenograft (PDX) model was employed to verify the therapeutic efficacy of NOB in vivo. RESULTS: We conducted functional studies and discovered that NOB inhibited the protective effect of autophagy via the PI3K/Akt/mTOR axis in GC cells. Based on previous research, we found that the overexpression of ACLY abrogated the NOB-induced autophagy-dependent cell death. In silico analysis predicted the formation of a stable complex between NOB and SREBP1. In vitro assays confirmed that NOB treatment increased the thermal stability of SREBP1 at the same temperature conditions. Moreover, CUT&TAG-qPCR analysis revealed that NOB could inhibit SREBP1 binding to the ACLY promoter. In the PDX model, NOB suppressed tumor growth, causing SREBP1 nuclear translocation inhibition, PI3K/Akt/mTOR inactivation, and autophagy-dependent cell death. CONCLUSION: NOB demonstrated the ability to directly bind to SREBP1, inhibiting its nuclear translocation and binding to the ACLY promoter, thereby inducing autophagy-dependent cell death via PI3K/Akt/mTOR pathway.

Dehydrocostus lactone suppresses gastric cancer progression by targeting ACLY to inhibit fatty acid synthesis and autophagic flux.

INTRODUCTION: Dehydrocostus lactone (Dehy), a natural sesquiterpene lactone from Saussurea lappa Clarke, displays remarkable efficacy in treating cancer and gastrointestinal disorders. However, its anti-gastric cancer (GC) effect remains poorly understood. OBJECTIVES: Our study aimed to elucidate the anti-GC effect of Dehy and its putative mechanism. METHODS: The anti-GC effect was assessed with MTT, colony formation, wound healing and transwell invasion assays. Cell apoptosis rate was detected by Annexin V-FITC/PI binding assay. Network pharmacology analysis and XF substrate oxidation stress test explored the underlying mechanism and altered metabolic phenotype. Lipogenic enzyme expressions and neutral lipid pool were measured to evaluate cellular lipid synthesis and storage. Biolayer interferometry and molecular docking investigated the direct target of Dehy. Autophagosomes were observed by transmission electron microscopy and MDC staining, while the autophagic flux was detected by mRFP-GFP-LC3 transfection. The clinical significance of ACLY was confirmed by tissue microarrays. Patient-derived xenograft (PDX) models were adopted to detect the clinical therapeutic potential of Dehy. RESULTS: Dehy prominently suppressed GC progression both in vitro and in vivo. Mechanistically, Dehy down-regulated the lipogenic enzyme ACLY, thereby reducing fatty acid synthesis and lipid reservation. Moreover, IKKß was identified as the direct target of Dehy. Dehy inhibited the phosphorylation of IKKß, promoting the ubiquitination and degradation of ACLY, thereby resulting in lipid depletion. Subsequently, GC cells initiated autophagy to replenish the missing lipids, whereas Dehy impeded this cytoprotective mechanism by down-regulating LAMP1 and LAMP2 expressions, which disrupted lysosomal membrane functions, ultimately leading to apoptosis. Additionally, Dehy exhibited potential in GC clinical therapy as it enhanced the efficacy of 5-Fluorouracil in PDX models. CONCLUSIONS: Our work identified Dehy as a desirable agent for blunting abnormal lipid metabolism and highlighted its inhibitory effect on protective autophagy, suggesting the future development of Dehy as a novel therapeutic drug for GC.

Recent advance of ATP citrate lyase inhibitors for the treatment of cancer and related diseases.

ATP citrate lyase (ACLY), a strategic metabolic enzyme that catalyzes the glycolytic to lipidic metabolism, has gained increasing attention as an attractive therapeutic target for hyperlipidemia, cancers and other human diseases. Despite of continual research efforts, targeting ACLY has been very challenging. In this field, most reported ACLY inhibitors are "substrate-like" analogues, which occupied with the same active pockets. Besides, some ACLY inhibitors have been disclosed through biochemical screening or high throughput virtual screening. In this review, we briefly summarized the cancer-related functions and the recent advance of ACLY inhibitors with a particular focus on the SAR studies and their modes of action. We hope to provide a timely and updated overview of ACLY and the discovery of new ACLY inhibitors.

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.

Metabolic dysregulation and emerging therapeutical targets for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an aggressive human cancer with increasing incidence worldwide. Multiple efforts have been made to explore pharmaceutical therapies to treat HCC, such as targeted tyrosine kinase inhibitors, immune based therapies and combination of chemotherapy. However, limitations exist in current strategies including chemoresistance for instance. Tumor initiation and progression is driven by reprogramming of metabolism, in particular during HCC development. Recently, metabolic associated fatty liver disease (MAFLD), a reappraisal of new nomenclature for non-alcoholic fatty liver disease (NAFLD), indicates growing appreciation of metabolism in the pathogenesis of liver disease, including HCC, thereby suggesting new strategies by targeting abnormal metabolism for HCC treatment. In this review, we introduce directions by highlighting the metabolic targets in glucose, fatty acid, amino acid and glutamine metabolism, which are suitable for HCC pharmaceutical intervention. We also summarize and discuss current pharmaceutical agents and studies targeting deregulated metabolism during HCC treatment. Furthermore, opportunities and challenges in the discovery and development of HCC therapy targeting metabolism are discussed.