Targeting acetate metabolism: Achilles' nightmare.

Recent advances in our understanding of tumour heterogeneity alongside studies investigating altered metabolism within transformed tissue have identified metabolic pathways critical to cancer cell survival. Leveraging this information presents a promising new avenue for the generation of cancer-specific therapeutics and improved patient outcomes.

Acetyl-CoA Synthetase 2 Promotes Cell Migration and Invasion of Renal Cell Carcinoma by Upregulating Lysosomal-Associated Membrane Protein 1 Expression.

BACKGROUND/AIMS: Reprogramming energy metabolism is an emerging hallmark of many cancers, and this alteration is especially evident in renal cell carcinomas (RCCs). However, few studies have been conducted on lipid metabolism. This study investigated the function and mechanism of lipid metabolism-related acetyl-CoA synthetase 2 (ACSS2) in RCC development, cell migration and invasion. METHODS: Quantitative real-time PCR (qRT-PCR) was used to determine the expression of ACSS2 in cancer tissue and adjacent tissue. The inhibition of ACSS2 expression was achieved by RNA interference, which was confirmed by qRT-PCR and Western blotting. Cell proliferation and apoptosis were detected by a CCK8 assay and a flow cytometry analysis, respectively. Cell migration and invasion were determined by the scratch and transwell assays. Following the knockdown of ACSS2 expression, the expression of the autophagy-related factor LAMP1 was measured by qRT-PCR and Western blotting. RESULTS: Compared to adjacent tissues, ACSS2 expression was upregulated in RCC cancer tissues and positively correlated with metastasis. Inhibition of ACSS2 had no effect on RCC cell proliferation or apoptosis. However, decreased ACSS2 expression was found to inhibit RCC cell migration and invasion. ACSS2 was determined to promote the expression of LAMP1, which can also promote cell migration. This pathway may be considered a potential mechanism through which ACSS2 participates in RCC development. CONCLUSION: These data suggest that ACSS2 is an important factor for promoting RCC development and is essential for cell migration and invasion, which it promotes by increasing the expression of LAMP1. Taken together, these findings reveal a potential target for the diagnosis and treatment of RCC.

Biochemical and kinetic characterization of the recombinant ADP-forming acetyl coenzyme A synthetase from the amitochondriate protozoan Entamoeba histolytica.

Entamoeba histolytica, an amitochondriate protozoan parasite that relies on glycolysis as a key pathway for ATP generation, has developed a unique extended PPi-dependent glycolytic pathway in which ADP-forming acetyl-coenzyme A (CoA) synthetase (ACD; acetate:CoA ligase [ADP-forming]; EC 6.2.1.13) converts acetyl-CoA to acetate to produce additional ATP and recycle CoA. We characterized the recombinant E. histolytica ACD and found that the enzyme is bidirectional, allowing it to potentially play a role in ATP production or in utilization of acetate. In the acetate-forming direction, acetyl-CoA was the preferred substrate and propionyl-CoA was used with lower efficiency. In the acetyl-CoA-forming direction, acetate was the preferred substrate, with a lower efficiency observed with propionate. The enzyme can utilize both ADP/ATP and GDP/GTP in the respective directions of the reaction. ATP and PPi were found to inhibit the acetate-forming direction of the reaction, with 50% inhibitory concentrations of 0.81 ± 0.17 mM (mean ± standard deviation) and 0.75 ± 0.20 mM, respectively, which are both in the range of their physiological concentrations. ATP and PPi displayed mixed inhibition versus each of the three substrates, acetyl-CoA, ADP, and phosphate. This is the first example of regulation of ACD enzymatic activity, and possible roles for this regulation are discussed.

Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes.

Acetyl-CoA produced in mitochondria from carbohydrate or amino acid catabolism needs to reach the cytosol to initiate de novo synthesis of fatty acids. All eukaryotes analyzed so far use a citrate/malate shuttle to transfer acetyl group equivalents from the mitochondrial matrix to the cytosol. Here we investigate how this acetyl group transfer occurs in the procyclic life cycle stage of Trypanosoma brucei, a protozoan parasite responsible of human sleeping sickness and economically important livestock diseases. Deletion of the potential citrate lyase gene, a critical cytosolic enzyme of the citrate/malate shuttle, has no effect on de novo biosynthesis of fatty acids from (14)C-labeled glucose, indicating that another route is used for acetyl group transfer. Because acetate is produced from acetyl-CoA in the mitochondrion of this parasite, we considered genes encoding cytosolic enzymes producing acetyl-CoA from acetate. We identified an acetyl-CoA synthetase gene encoding a cytosolic enzyme (AceCS), which is essential for cell viability. Repression of AceCS by inducible RNAi results in a 20-fold reduction of (14)C-incorporation from radiolabeled glucose or acetate into de novo synthesized fatty acids. Thus, we demonstrate that the essential cytosolic enzyme AceCS of T. brucei is responsible for activation of acetate into acetyl-CoA to feed de novo biosynthesis of lipids. To date, Trypanosoma is the only known eukaryotic organism that uses acetate instead of citrate to transfer acetyl groups over the mitochondrial membrane for cytosolic lipid synthesis.

Chemogenomics identifies acetyl-coenzyme A synthetase as a target for malaria treatment and prevention.

We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression.

Structural Characterization of the Reaction and Substrate Specificity Mechanisms of Pathogenic Fungal Acetyl-CoA Synthetases.

Acetyl CoA synthetases (ACSs) are Acyl-CoA/NRPS/Luciferase (ANL) superfamily enzymes that couple acetate with CoA to generate acetyl CoA, a key component of central carbon metabolism in eukaryotes and prokaryotes. Normal mammalian cells are not dependent on ACSs, while tumor cells, fungi, and parasites rely on acetate as a precursor for acetyl CoA. Consequently, ACSs have emerged as a potential drug target. As part of a program to develop antifungal ACS inhibitors, we characterized fungal ACSs from five diverse human fungal pathogens using biochemical and structural studies. ACSs catalyze a two-step reaction involving adenylation of acetate followed by thioesterification with CoA. Our structural studies captured each step of these two half-reactions including the acetyl-adenylate intermediate of the first half-reaction in both the adenylation conformation and the thioesterification conformation and thus provide a detailed picture of the reaction mechanism. We also used a systematic series of increasingly larger alkyl adenosine esters as chemical probes to characterize the structural basis of the exquisite ACS specificity for acetate over larger carboxylic acid substrates. Consistent with previous biochemical and genetic data for other enzymes, structures of fungal ACSs with these probes bound show that a key tryptophan residue limits the size of the alkyl binding site and forces larger alkyl chains to adopt high energy conformers, disfavoring their efficient binding. Together, our analysis provides highly detailed structural models for both the reaction mechanism and substrate specificity that should be useful in designing selective inhibitors of eukaryotic ACSs as potential anticancer, antifungal, and antiparasitic drugs.

Targeting ACSS2 with a Transition-State Mimetic Inhibits Triple-Negative Breast Cancer Growth.

Acetyl-CoA is a vitally important and versatile metabolite used for many cellular processes including fatty acid synthesis, ATP production, and protein acetylation. Recent studies have shown that cancer cells upregulate acetyl-CoA synthetase 2 (ACSS2), an enzyme that converts acetate to acetyl-CoA, in response to stresses such as low nutrient availability and hypoxia. Stressed cancer cells use ACSS2 as a means to exploit acetate as an alternative nutrient source. Genetic depletion of ACSS2 in tumors inhibits the growth of a wide variety of cancers. However, there are no studies on the use of an ACSS2 inhibitor to block tumor growth. In this study, we synthesized a small-molecule inhibitor that acts as a transition-state mimetic to block ACSS2 activity in vitro and in vivo. Pharmacologic inhibition of ACSS2 as a single agent impaired breast tumor growth. Collectively, our findings suggest that targeting ACSS2 may be an effective therapeutic approach for the treatment of patients with breast cancer. SIGNIFICANCE: These findings suggest that targeting acetate metabolism through ACSS2 inhibitors has the potential to safely and effectively treat a wide range of patients with cancer.

Acetate Recapturing by Nuclear Acetyl-CoA Synthetase 2 Prevents Loss of Histone Acetylation during Oxygen and Serum Limitation.

Acetyl-CoA is a key metabolic intermediate with an important role in transcriptional regulation. The nuclear-cytosolic acetyl-CoA synthetase 2 (ACSS2) was found to sustain the growth of hypoxic tumor cells. It generates acetyl-CoA from acetate, but exactly which pathways it supports is not fully understood. Here, quantitative analysis of acetate metabolism reveals that ACSS2 fulfills distinct functions depending on its cellular location. Exogenous acetate uptake is controlled by expression of both ACSS2 and the mitochondrial ACSS1, and ACSS2 supports lipogenesis. The mitochondrial and lipogenic demand for two-carbon acetyl units considerably exceeds the uptake of exogenous acetate, leaving it to only sparingly contribute to histone acetylation. Surprisingly, oxygen and serum limitation increase nuclear localization of ACSS2. We find that nuclear ACSS2 recaptures acetate released from histone deacetylation for recycling by histone acetyltransferases. Our work provides evidence for limited equilibration between nuclear and cytosolic acetyl-CoA and demonstrates that ACSS2 retains acetate to maintain histone acetylation.

Connection of propionyl-CoA metabolism to polyketide biosynthesis in Aspergillus nidulans.

Propionyl-CoA is an intermediate metabolite produced through a variety of pathways including thioesterification of propionate and catabolism of odd chain fatty acids and select amino acids. Previously, we found that disruption of the methylcitrate synthase gene, mcsA, which blocks propionyl-CoA utilization, as well as growth on propionate impaired production of several polyketides-molecules typically derived from acetyl-CoA and malonyl-CoA-including sterigmatocystin (ST), a potent carcinogen, and the conidiospore pigment. Here we describe three lines of evidence that demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis. First, inactivation of a putative propionyl-CoA synthase, PcsA, which converts propionate to propionyl-CoA, restored polyketide production and reduced cellular propionyl-CoA content in a DeltamcsA background. Second, inactivation of the acetyl-CoA synthase, FacA, which is also involved in propionate utilization, restored polyketide production in the DeltamcsA background. Third, fungal growth on several compounds (e.g., heptadecanoic acid, isoleucine, and methionine) whose catabolism includes the formation of propionyl-CoA, were found to inhibit ST and conidiospore pigment production. These results demonstrate that excessive propionyl-CoA levels in the cell can inhibit polyketide synthesis.

Allicin, a naturally occurring antibiotic from garlic, specifically inhibits acetyl-CoA synthetase.

Allicin is shown to be a specific inhibitor of the acetyl-CoA synthetases from plants, yeast and mammals. The bacterial acetyl-CoA-forming system, consisting of acetate kinase and phosphotransacetylase, was inhibited too. Non-specific interaction with sulfhydryl-groups could be excluded in experiments with dithioerythritol and p-hydroxymercuribenzoate. Binding of allicin to the enzyme is non-covalent and reversible. [14C]-Acetate incorporation into fatty acids of isolated plastids was inhibited by allicin with an I50-value lower than 10 microM. Other enzymes of the fatty acid synthesis sequence were not affected, as was shown using precursors other than acetate.

A homogeneous scintillation proximity format for monitoring the activity of recombinant human long-chain-fatty-acyl-CoA synthetase 5.

Fatty acyl coenzyme A (CoA) synthetases are a group of enzymes responsible for the activation of fatty acids through ligated high-energy CoA thioester bonds. Ultimately these fatty acyl-CoA conjugates are routed toward either anabolic or catabolic pathways. Long-chain-fatty-acid-CoA ligase 5 (LACS 5) utilizes a wide range of saturated fatty acids with a substrate preference for C16-C18 unsaturated fatty acids. This enzyme represents a new class of potential drug targets, and, hence, our efforts were focused upon developing a robust assay for utilization in a high throughput screen. Toward that end, we describe a radiometric homogeneous measurement of the enzymatic reaction by employing ionic capture of the reaction product onto YSi scintillation proximity assay (SPA) beads. We present kinetic and inhibition data for LACS 5 using this SPA format. Our results show that the assay method is both robust and well suited for this class of lipid-metabolizing enzymes.

Evidence to suggest that extracellular acetate is accumulated by rat hippocampal cholinergic nerve terminals for acetylcholine formation and release.

It is well established that extracellular choline is transported into central cholinergic nerve terminals by 'high' and 'low' affinity processes to form the neurotransmitter acetylcholine (ACh). The intent of the present investigation was to ascertain whether extracellular acetate might also be transported into central cholinergic nerve terminals to form ACh. To test this possibility, rat hippocampal tissue was incubated with varying concentrations of extracellular [1-(14)C]acetate (0.1-100 microM) and the uptake of [1-(14)C]acetate and the amount of [14C]ACh formed by the tissue determined. The results indicated that the uptake of extracellular [1-(14)C]acetate was temperature-dependent and saturable having an apparent Michaelis constant (Km) of 22 microM. The formation of [14C]ACh in the tissue as a function of extracellular [1-(14)C]acetate appeared to occur by both 'high' and 'low' affinity processes with apparent Km values of 0.5 and 19.6 microM, respectively. In other experiments, three inhibitors (lithium, allicin and sodium) of acetyl CoA synthetase (EC 6.2.1.1 acetate: CoA ligase), the enzyme which converts acetate to acetyl CoA when ATP and CoA are present, inhibited [1-(14)C]acetate uptake and the amount of [14C]ACh formed from that [1-(14)C]acetate. Additionally, vesamicol, an inhibitor of ACh transport into synaptic vesicles, blocked the filling of a synaptic vesicle-enriched fraction of hippocampal tissue with newly synthesized [14C]ACh formed from extracellular [1-(14)C]acetate. High K+ depolarization of hippocampal tissue loaded with extracellular [1-(14)C]acetate not only increased the synthesis but also the release of [14C]ACh. These results suggest that extracellular acetate is recycled by rat hippocampal cholinergic nerve terminals for the formation and release of ACh. They also suggest that the enzyme acetyl CoA synthetase mediates extracellular acetate uptake into hippocampal cholinergic nerve terminals by metabolizing it to acetyl CoA and thereby creating a diffusion gradient for it to follow.

Stable analogs of acyl adenylates. Inhibition of acetyl- and acyl-CoA synthetase by adenosine 5'-alkylphosphates.

Adenosine 5'-alkylphosphates are potent inhibitors of acetyl- and acyl-CoA synthetase. In each case, the most effective inhibitor in the series is homologous with the tightly bound acyl adenylate intermediate. Adenosine 5'-ethylphosphate (Ki = 33 nM) is 88-fold more potent than adenosine 5'-methylphosphate (Ki = 2900 nM) as a competitive inhibitor of acetyl-CoA synthetase; the contribution of a single carbon to the observed binding energy (-11 kJ/mol) is much larger than is typically observed.

Effects of imide analogs on enzymes required for cholesterol and fatty acid synthesis.

Twelve imide analogs were examined for their ability to lower serum cholesterol and triglyceride levels in mice. Potent activity was observed for compounds containing a phthalimide or saccharin ring structure. The ability to lower serum cholesterol appears to be related to the ability to suppress acetyl-CoA synthetase activity. The availability of acetyl-CoA in the cytoplasm is a key regulatory component for cholesterol and fatty acid synthesis. The capacity to reduce serum triglycerides was related directly to the ability of the compound to inhibit acetyl-CoA carboxylase activity, the regulatory enzyme of fatty acid synthesis.

Antihyperlipidemic activity of amine cyanoboranes, amine carboxyboranes, and related compounds.

A series of amine cyanoboranes and amine carboxyboranes were observed to be antihyperlipidemic agents in mice; i.e., they lowered serum cholesterol and triglyceride levels significantly. These compounds appeared to inhibit lipid synthesis in the early stages. The ability to lower serum cholesterol levels appeared to correlate with the suppression of the regulatory enzyme of cholesterol synthesis, beta-hydroxy-beta-methylglutaryl-CoA reductase activity. The reduction of serum triglycerides correlated with ability of the borane compound to suppress liver fatty acid synthetase activity.

[Organophosphate analogs of biologically active compounds. XIV. Halophosphonate analogs of ATP as acetyl CoA carboxylase inhibitors]. / Fosfororganicheskie analogi biologicheski aktivnykh soedinenii. XIV. Galoidfosfonatnye analogi ATP kak ingibitory Ac CoA-karboksilazy.

Halophosphonate ATP analogues pp[CHBr]pA and p[CHBr]ppA synthesised from bromomethylenebisphosphonate and adenosine derivatives, proved to be effective competitive inhibitors of Ac-CoA-carboxylase (CE 6.4.1.2) from rat liver (Ki = 0,2 mM). The inhibitory effects of both analogues were reversible and higher than those of some other ATP analogues. Another enzyme, Ac-CoA-synthetase (CE 6.2.1.1), with a different mode of ATP-cleavage showed wider specificity to ATP-analogues than Ac-CoA-carboxylase.

[Regulation of acetate metabolism in a strain of Acinetobacter sp., growing on ethanol]. / Reguliatsiia metabolizma atsetata u shtamma Acinetobacter sp., rastushchego na étanole.

Ethanol metabolism in Acinetobacter sp. is limited by the rate of acetate assimilation in a reaction catalyzed by acetyl-CoA synthetase (EC 6.2.1.1). Effects of ions (sodium, potassium, and magnesium), byproducts of ethanol and acetaldehyde oxidation (NADH and NADPH), and pantothenic acid on this enzyme have been studied (sodium, NADH, and NADPH inhibit acetyl-CoA synthetase; pantothenic acid, potassium, and magnesium act as the enzyme activators). Conditions of culturing were developed, under which ethanol, acetaldehyde, and acetate in Acinetobacter cells were oxidized at the same rates, producing a threefold increase in the activity of acetyl-CoA synthetase in the cell-free extract. The results of studies of acetyl-CoA synthetase regulation in a mutant strain of Acinetobacter sp., which is incapable of forming exopolysaccharides, provide a basis for refining the technology of ethapolan production, involving the use of C2 substrates.

Inhibition effect of isopropanol on acetyl-CoA synthetase expression level of acetoclastic methanogen, Methanosaeta concilii.

Isopropanol is a widely found solvent in industrial wastewaters, which have commonly been treated using anaerobic systems. In this study, inhibitory effect of isopropanol on the key microbial group in anaerobic bioreactors, acetoclastic methanogens, was investigated. Anaerobic sludges in serum bottles were repeatedly fed with acetate and isopropanol; and quantitative real-time PCR was used for determining effect of isopropanol on the expression level of a key enzyme in acetoclastic methane production, acetyl-CoA synthetase of Methanosaeta concilii. Active Methanosaeta spp. cells were also quantified using Fluorescent in situ hybridization (FISH). Transcript abundance of acetyl-CoA synthetase was 1.23±0.62×10(6) mRNAs/mL in the uninhibited reactors with 222 mL cumulative methane production. First exposure to isopropanol resulted in 71.2%, 84.7%, 89.2% and 94.6% decrease in mRNA level and 35.0%, 65.0%, 91.5% and 100.0% reduction in methane production for isopropanol concentrations of 0.1 M, 0.5 M, 1.0 M and 2.0 M, respectively. Repeated exposures resulted in higher inhibitions; and at the end of test, fluorescent intensities of active Methanosaeta cells were significantly decreased due to isopropanol. The overall results indicated that isopropanol has an inhibitory effect on acetoclastic methanogenesis; and the inhibition can be detected by monitoring level of acetyl-CoA transcripts and rRNA level.

[Study of acetyl-CoA-synthetase from staphylococcus aureus]. / Issledovanie Atsetil-koa-Cintetazy is Solotistogo Stafilokokka

Acetyl-CoA-synthetase was isolated from cells of St. aureus 209-P. The method of isolation and partial purification of the enzyme is worked out. Km values of the enzyme for acetate, CoA and ATP are calculated. p-Chloromercuribenzoate and monoiodoacetate were shown to inhibit the enzyme activity. The enzyme activity is estimated depending on the age of the cell culture and on the presence of acetate in the culture medium.