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.

The structure of succinyl-CoA synthetase bound to the succinyl-phosphate intermediate clarifies the catalytic mechanism of ATP-citrate lyase.

Succinyl-CoA synthetase (SCS) catalyzes a three-step reaction in the citric acid cycle with succinyl-phosphate proposed as a catalytic intermediate. However, there are no structural data to show the binding of succinyl-phosphate to SCS. Recently, the catalytic mechanism underlying acetyl-CoA production by ATP-citrate lyase (ACLY) has been debated. The enzyme belongs to the family of acyl-CoA synthetases (nucleoside diphosphate-forming) for which SCS is the prototype. It was postulated that the amino-terminal portion catalyzes the full reaction and the carboxy-terminal portion plays only an allosteric role. This interpretation was based on the partial loss of the catalytic activity of ACLY when Glu599 was mutated to Gln or Ala, and on the interpretation that the phospho-citryl-CoA intermediate was trapped in the 2.85 Šresolution structure from cryogenic electron microscopy (cryo-EM). To better resolve the structure of the intermediate bound to the E599Q mutant, the equivalent mutation, E105αQ, was made in human GTP-specific SCS. The structure of the E105αQ mutant shows succinyl-phosphate bound to the enzyme at 1.58 Šresolution when the mutant, after phosphorylation in solution by Mg2+-ATP, was crystallized in the presence of magnesium ions, succinate and desulfo-CoA. The E105αQ mutant is still active but has a specific activity that is 120-fold less than that of the wild-type enzyme, with apparent Michaelis constants for succinate and CoA that are 50-fold and 11-fold higher, respectively. Based on this high-resolution structure, the cryo-EM maps of the E599Q ACLY complex reported previously should have revealed the binding of citryl-phosphate and CoA and not phospho-citryl-CoA.

Discovery of a Novel Macrocyclic ATP Citrate Lyase Inhibitor.

ATP citrate lyase (ACLY) is an important metabolic enzyme involved in the synthesis of fatty acid and cholesterol. The inhibition of ACLY is considered as a promising therapeutic strategy for various metabolic diseases and numerous malignancies. In this study, a novel macrocyclic compound 2 has been identified as a potent ACLY inhibitor with the "ring closing" strategy for conformational restriction based on NDI-091143. It showed potent ACLY inhibitory activity and binding affinity comparable to the positive control. Furthermore, compared with the positive control (T1/2 = 3.36 min), the metabolic stability of 2 in HLMs (T1/2 = 531.22 min) was significantly improved. All of these results characterized 2 as a promising lead compound worthy of further study.

ATP citrate lyase: A central metabolic enzyme in cancer.

ACLY links energy metabolism provided by catabolic pathways to biosynthesis. ACLY, which has been found to be overexpressed in many cancers, converts citrate into acetyl-CoA and OAA. The first of these molecules supports protein acetylation, in particular that of histone, and de novo lipid synthesis, and the last one sustains the production of aspartate (required for nucleotide and polyamine synthesis) and the regeneration of NADPH,H+(consumed in redox reaction and biosynthesis). ACLY transcription is promoted by SREBP1, its activity is stabilized by acetylation and promoted by AKT phosphorylation (stimulated by growth factors and glucose abundance). ACLY plays a pivotal role in cancer metabolism through the potential deprivation of cytosolic citrate, a process promoting glycolysis through the enhancement of the activities of PFK 1 and 2 with concomitant activation of oncogenic drivers such as PI3K/AKT which activate ACLY and the Warburg effect in a feed-back loop. Pending the development of specific inhibitors and tailored methods for identifying which specific metabolism is involved in the development of each tumor, ACLY could be targeted by inhibitors such as hydroxycitrate and bempedoic acid. The administration of citrate at high level mimics a strong inhibition of ACLY and could be tested to strengthen the effects of current therapies.

Molecular basis for acetyl-CoA production by ATP-citrate lyase.

ATP-citrate lyase (ACLY) synthesizes cytosolic acetyl coenzyme A (acetyl-CoA), a fundamental cellular building block. Accordingly, aberrant ACLY activity is observed in many diseases. Here we report cryo-EM structures of human ACLY, alone or bound to substrates or products. ACLY forms a homotetramer with a rigid citrate synthase homology (CSH) module, flanked by four flexible acetyl-CoA synthetase homology (ASH) domains; CoA is bound at the CSH-ASH interface in mutually exclusive productive or unproductive conformations. The structure of a catalytic mutant of ACLY in the presence of ATP, citrate and CoA substrates reveals a phospho-citryl-CoA intermediate in the ASH domain. ACLY with acetyl-CoA and oxaloacetate products shows the products bound in the ASH domain, with an additional oxaloacetate in the CSH domain, which could function in ACLY autoinhibition. These structures, which are supported by biochemical and biophysical data, challenge previous proposals of the ACLY catalytic mechanism and suggest additional therapeutic possibilities for ACLY-associated metabolic disorders.

Identification of the active site residues in ATP-citrate lyase's carboxy-terminal portion.

ATP-citrate lyase (ACLY) catalyzes production of acetyl-CoA and oxaloacetate from CoA and citrate using ATP. In humans, this cytoplasmic enzyme connects energy metabolism from carbohydrates to the production of lipids. In certain bacteria, ACLY is used to fix carbon in the reductive tricarboxylic acid cycle. The carboxy(C)-terminal portion of ACLY shows sequence similarity to citrate synthase of the tricarboxylic acid cycle. To investigate the roles of residues of ACLY equivalent to active site residues of citrate synthase, these residues in ACLY from Chlorobium limicola were mutated, and the proteins were investigated using kinetics assays and biophysical techniques. To obtain the crystal structure of the C-terminal portion of ACLY, full-length C. limicola ACLY was cleaved, first non-specifically with chymotrypsin and subsequently with Tobacco Etch Virus protease. Crystals of the C-terminal portion diffracted to high resolution, providing structures that show the positions of active site residues and how ACLY tetramerizes.

Computational de-orphanization of the olive oil biophenol oleacein: Discovery of new metabolic and epigenetic targets.

The health promoting effects of extra virgin olive oil (EVOO) relate to its unique repertoire of phenolic compounds. Here, we used a chemoinformatics approach to computationally identify endogenous ligands and assign putative biomolecular targets to oleacein, one of the most abundant secoiridoids in EVOO. Using a structure-based virtual profiling software tool and reference databases containing more than 9000 binding sites protein cavities, we identified 996 putative oleacein targets involving more than 700 proteins. We subsequently identified the high-level functions of oleacein in terms of biomolecular interactions, signaling pathways, and protein-protein interaction (PPI) networks. Delineation of the oleacein target landscape revealed that the most significant modules affected by oleacein were associated with metabolic processes (e.g., glucose and lipid metabolism) and chromatin-modifying enzymatic activities (i.e., histone post-translational modifications). We experimentally confirmed that, in a low-micromolar physiological range (<20 µmol/l), oleacein was capable of inhibiting the catalytic activities of predicted metabolic and epigenetic targets including nicotinamide N-methyltransferase, ATP-citrate lyase, lysine-specific demethylase 6A, and N-methyltransferase 4. Our computational de-orphanization of oleacein provides new mechanisms through which EVOO biophenols might operate as chemical prototypes capable of modulating the biologic machinery of healthy aging.

An allosteric mechanism for potent inhibition of human ATP-citrate lyase.

ATP-citrate lyase (ACLY) is a central metabolic enzyme and catalyses the ATP-dependent conversion of citrate and coenzyme A (CoA) to oxaloacetate and acetyl-CoA1-5. The acetyl-CoA product is crucial for the metabolism of fatty acids6,7, the biosynthesis of cholesterol8, and the acetylation and prenylation of proteins9,10. There has been considerable interest in ACLY as a target for anti-cancer drugs, because many cancer cells depend on its activity for proliferation2,5,11. ACLY is also a target against dyslipidaemia and hepatic steatosis, with a compound currently in phase 3 clinical trials4,5. Many inhibitors of ACLY have been reported, but most of them have weak activity5. Here we report the development of a series of low nanomolar, small-molecule inhibitors of human ACLY. We have also determined the structure of the full-length human ACLY homo-tetramer in complex with one of these inhibitors (NDI-091143) by cryo-electron microscopy, which reveals an unexpected mechanism of inhibition. The compound is located in an allosteric, mostly hydrophobic cavity next to the citrate-binding site, and requires extensive conformational changes in the enzyme that indirectly disrupt citrate binding. The observed binding mode is supported by and explains the structure-activity relationships of these compounds. This allosteric site greatly enhances the 'druggability' of ACLY and represents an attractive target for the development of new ACLY inhibitors.

Structure of ATP citrate lyase and the origin of citrate synthase in the Krebs cycle.

Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine2, and the acetylation of histones and proteins3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue1 and in cholinergic neurons2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer8-11 and hyperlipidaemia12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth.

ATP-citrate lyase multimerization is required for coenzyme-A substrate binding and catalysis.

ATP-citrate lyase (ACLY) is a major source of nucleocytosolic acetyl-CoA, a fundamental building block of carbon metabolism in eukaryotes. ACLY is aberrantly regulated in many cancers, cardiovascular disease, and metabolic disorders. However, the molecular mechanisms determining ACLY activity and function are unclear. To this end, we investigated the role of the uncharacterized ACLY C-terminal citrate synthase homology domain in the mechanism of acetyl-CoA formation. Using recombinant, purified ACLY and a suite of biochemical and biophysical approaches, including analytical ultracentrifugation, dynamic light scattering, and thermal stability assays, we demonstrated that the C terminus maintains ACLY tetramerization, a conserved and essential quaternary structure in vitro and likely also in vivo Furthermore, we show that the C terminus, only in the context of the full-length enzyme, is necessary for full ACLY binding to CoA. Together, we demonstrate that ACLY forms a homotetramer through the C terminus to facilitate CoA binding and acetyl-CoA production. Our findings highlight a novel and unique role of the C-terminal citrate synthase homology domain in ACLY function and catalysis, adding to the understanding of the molecular basis for acetyl-CoA synthesis by ACLY. This newly discovered means of ACLY regulation has implications for the development of novel ACLY modulators to target acetyl-CoA-dependent cellular processes for potential therapeutic use.

Binding of hydroxycitrate to human ATP-citrate lyase.

Hydroxycitrate from the fruit of Garcinia cambogia [i.e. (2S,3S)-2-hydroxycitrate] is the best-known inhibitor of ATP-citrate lyase. Well diffracting crystals showing how the inhibitor binds to human ATP-citrate lyase were grown by modifying the protein. The protein was modified by introducing cleavage sites for Tobacco etch virus protease on either side of a disordered linker. The protein crystallized consisted of residues 2-425-ENLYFQ and S-488-810 of human ATP-citrate lyase. (2S,3S)-2-Hydroxycitrate binds in the same orientation as citrate, but the citrate-binding domain (residues 248-421) adopts a different orientation with respect to the rest of the protein (residues 4-247, 490-746 and 748-809) from that previously seen. For the first time, electron density was evident for the loop that contains His760, which is phosphorylated as part of the catalytic mechanism. The pro-S carboxylate of (2S,3S)-2-hydroxycitrate is available to accept a phosphoryl group from His760. However, when co-crystals were grown with ATP and magnesium ions as well as either the inhibitor or citrate, Mg2+-ADP was bound and His760 was phosphorylated. The phosphoryl group was not transferred to the organic acid. This led to the interpretation that the active site is trapped in an open conformation. The strategy of designing cleavage sites to remove disordered residues could be useful in determining the crystal structures of other proteins.

Design and synthesis of emodin derivatives as novel inhibitors of ATP-citrate lyase.

Aberrant cellular metabolism drives cancer proliferation and metastasis. ATP citrate lyase (ACL) plays a critical role in generating cytosolic acetyl CoA, a key building block for de novo fatty acid and cholesterol biosynthesis. ACL is overexpressed in cancer cells, and siRNA knockdown of ACL limits cancer cell proliferation and reduces cancer stemness. We characterized a new class of ACL inhibitors bearing the key structural feature of the natural product emodin. Structure-activity relationship (SAR) study led to the identification of 1d as a potent lead that demonstrated dose-dependent inhibition of proliferation and cancer stemness of the A549 lung cancer cell line. Computational modeling indicates this class of inhibitors occupies an allosteric binding site and blocks the entrance of the substrate citrate to its binding site.

Molecular cloning and characterization of the ATP citrate lyase from carotenogenic yeast Phaffia rhodozyma.

ATP citrate lyase (ACL), is a key cytosolic source of acetyl-CoA for fatty acid and sterol biosynthesis and appear to be involved in carotenoid biosynthesis in yeasts. Three homologous DNA sequences encoding ACLs in Phaffia rhodozyma were isolated i.e two genes and one cDNA. The two genes were multi-intronic, with 3450-bp-coding sequences and both genes, as the cDNA, encoded identical 120.1-kDa polypeptides. Full-length amino acid sequences of these ACLs showed the two multidomains, PLN02235 and PLN02522, which are necessary for activity. The ACLs showed 82-87% similarity to putative ACLs from other basidiomycetes and 71% similarity to human ACL. The acl cDNA was used to express the heterologous ACL 6XHis-tagged which was identified using MALDI-TOF-MS. The sequenced peptides with 42.2% coverage showed 100% identity to the amino acid sequence generated in silico. The recombinant ACL purified to homogeneity showed an activity of 2 U. This is the first study to characterize a recombinant ACL from a carotenogenic yeast. The present study provides a key foundation for future studies to assess (a) the possible occurrence of alternative splicing, (b) identify the promoter(s) sequence(s) and (c) the involvement of ACL in the differential regulation of fatty acid and carotenoid biosynthesis in yeasts.

Phosphoserine Lyase Deoxyribozymes: DNA-Catalyzed Formation of Dehydroalanine Residues in Peptides.

Dehydroalanine (Dha) is a nonproteinogenic electrophilic amino acid that is a synthetic intermediate or product in the biosynthesis of several bioactive cyclic peptides such as lantibiotics, thiopeptides, and microcystins. Dha also enables labeling of proteins and synthesis of post-translationally modified proteins and their analogues. However, current chemical approaches to introducing Dha into peptides have substantial limitations. Using in vitro selection, here we show that DNA can catalyze Zn(2+) or Zn(2+)/Mn(2+)-dependent formation of Dha from phosphoserine (pSer), i.e., exhibit pSer lyase activity, a fundamentally new DNA-catalyzed reaction. Two new pSer lyase deoxyribozymes, named Dha-forming deoxyribozymes 1 and 2 (DhaDz1 and DhaDz2), each function with multiple turnover on the model hexapeptide substrate that was used during selection. Using DhaDz1, we generated Dha from pSer within an unrelated linear 13-mer peptide. Subsequent base-promoted intramolecular cyclization of homocysteine into Dha formed a stable cystathionine (thioether) analogue of the complement inhibitor compstatin. These findings establish the fundamental catalytic ability of DNA to eliminate phosphate from pSer to form Dha and suggest that with further development, pSer lyase deoxyribozymes will have broad practical utility for site-specific enzymatic synthesis of Dha from pSer in peptide substrates.

Genome-wide identification of citrus ATP-citrate lyase genes and their transcript analysis in fruits reveals their possible role in citrate utilization.

ATP-citrate lyase (ACL, EC4.1.3.8) catalyzes citrate to oxaloacetate and acetyl-CoA in the cell cytosol, and has important roles in normal plant growth and in the biosynthesis of some secondary metabolites. We identified three ACL genes, CitACLα1, CitACLα2, and CitACLß1, in the citrus genome database. Both CitACLα1 and CitACLα2 encode putative ACL α subunits with 82.5 % amino acid identity, whereas CitACLß1 encodes a putative ACL ß subunit. Gene structure analysis showed that CitACLα1 and CitACLα2 had 12 exons and 11 introns, and CitACLß1 had 16 exons and 15 introns. CitACLα1 and CitACLß1 were predominantly expressed in flower, and CitACLα2 was predominantly expressed in stem and fibrous roots. As fruits ripen, the transcript levels of CitACLα1, CitACLß1, and/or CitACLα2 in cultivars 'Niuher' and 'Owari' increased, accompanied by significant decreases in citrate content, while their transcript levels decreased significantly in 'Egan No. 1' and 'Iyokan', although citrate content also decreased. In 'HB pummelo', in which acid content increased as fruit ripened, and in acid-free pummelo, transcript levels of CitACLα2, CitACLß1, and/or CitACLα1 increased. Moreover, mild drought stress and ABA treatment significantly increased citrate contents in fruits. Transcript levels of the three genes were significantly reduced by mild drought stress, and the transcript level of only CitACLß1 was significantly reduced by ABA treatment. Taken together, these data indicate that the effects of ACL on citrate use during fruit ripening depends on the cultivar, and the reduction in ACL gene expression may be attributed to citrate increases under mild drought stress or ABA treatment.

ATP citrate lyase (ACLY): a promising target for cancer prevention and treatment.

ATP citrate lyase (ACLY), an important enzyme involved in lipid biogenesis linked with glucose metabolism, catalyzes the conversion of citrate to oxaloacetic acid (OAA) and acetyl-CoA. The obtained acetyl-CoA is required for lipid synthesis during membrane biogenesis, as well as for histone acetylation reactions to regulate the expression of certain proteins in aberrantly proliferating cancer cells. Studies have shown a role for ACLY in tumorigenesis whereby increased levels of the enzyme leads to increased metabolic activity via activation of Akt signaling. Increasing lines of evidence suggest that enzymes involved in lipid biogenesis play a significant role in cancer cell proliferation and progression. In many cancer types such as glioblastoma, colorectal cancer, breast cancer, non-small cell lung cancer, hepatocellular carcinoma etc., the level of ACLY has been found to be quite high as compared to normal cells. Cancer cell growth related to overexpression of ACLY can be inhibited by using chemical inhibitors or by the knockdown of ACLY gene. Inhibition of ACLY leads to changes in cancer cell metabolism that promotes tumor growth and proliferation. This review summarizes the role of ACLY in cancer development and its inhibitors in cancer treatment.

Acetylation stabilizes ATP-citrate lyase to promote lipid biosynthesis and tumor growth.

Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.

On the catalytic mechanism of human ATP citrate lyase.

ATP citrate lyase (ACL) catalyzes an ATP-dependent biosynthetic reaction which produces acetyl-coenzyme A and oxaloacetate from citrate and coenzyme A (CoA). Studies were performed with recombinant human ACL to ascertain the nature of the catalytic phosphorylation that initiates the ACL reaction and the identity of the active site residues involved. Inactivation of ACL by treatment with diethylpyrocarbonate suggested the catalytic role of an active site histidine (i.e., His760), which was proposed to form a phosphohistidine species during catalysis. The pH-dependence of the pre-steady-state phosphorylation of ACL with [γ-(33)P]-ATP revealed an ionizable group with a pK(a) value of ~7.5, which must be unprotonated for the catalytic phosphorylation of ACL to occur. Mutagenesis of His760 to an alanine results in inactivation of the biosynthetic reaction of ACL, in good agreement with the involvement of a catalytic histidine. The nature of the formation of the phospho-ACL was further investigated by positional isotope exchange using [γ-(18)O(4)]-ATP. The ß,γ-bridge to nonbridge positional isotope exchange rate of [γ-(18)O(4)]-ATP achieved its maximal rate of 14 s(-1) in the absence of citrate and CoA. This rate decreased to 5 s(-1) when citrate was added, and was found to be 10 s(-1) when both citrate and CoA were present. The rapid positional isotope exchange rates indicated the presence of one or more catalytically relevant, highly reversible phosphorylated intermediates. Steady-state measurements in the absence of citrate and CoA showed that MgADP was produced by both wild type and H760A forms of ACL, with rates at three magnitudes lower than that of k(cat) for the full biosynthetic reaction. The ATPase activity of ACL, along with the small yet significant positional isotope exchange rate observed in H760A mutant ACL (~150 fold less than wild type), collectively suggested the presence of a second, albeit unproductive, phosphoryl transfer in ACL. Mathematical analysis and computational simulation suggested that the desorption of MgADP at a rate of ~7 s(-1) was the rate-limiting step in the biosynthesis of AcCoA and oxaloacetate.

ATP-citrate lyase: a mini-review.

ATP-citrate lyase (ACLY) is a cytosolic enzyme that catalyzes generation of acetyl-CoA from citrate. Acetyl-CoA is a vital building block for the endogenous biosynthesis of fatty acids and cholesterol and is involved in isoprenoid-based protein modifications. Acetyl-CoA is also required for acetylation reactions that modify proteins such as histone acetylation. In the present review some of the known features of ACLY such as tissue distribution, subcellular localization, enzymatic properties, gene regulation and associated physiological conditions are highlighted.

ATP citrate lyase inhibitors as novel cancer therapeutic agents.

ATP citrate lyase (ACL or ACLY) is an extra-mitochondrial enzyme widely distributed in various human and animal tissues. ACL links glucose and lipid metabolism by catalyzing the formation of acetyl-CoA and oxaloacetate from citrate produced by glycolysis in the presence of ATP and CoA. ACL is aberrantly expressed in many immortalized cells and tumors, such as breast, liver, colon, lung and prostate cancers, and is correlated reversely with tumor stage and differentiation, serving as a negative prognostic marker. ACL is an upstream enzyme of the long chain fatty acid synthesis, providing acetyl-CoA as an essential component of the fatty acid synthesis. Therefore, ACL is a key enzyme of cellular lipogenesis and potent target for cancer therapy. As a hypolipidemic strategy of metabolic syndrome and cancer treatment, many small chemicals targeting ACL have been designed and developed. This review article provides an update for the research and development of ACL inhibitors with a focus on their patent status, offering a new insight into their potential application.