Discovery of furan carboxylate derivatives as novel inhibitors of ATP-citrate lyase via virtual high-throughput screening

The enzyme ATP citrate lyase (ACL) catalyzes the formation of cytosolic acetyl CoA, the starting material for de novo lipid and cholesterol biosynthesis. The dysfunction and upregulation of ACL in numerous cancers makes it an attractive target for developing anticancer therapies. ACL inhibition by shRNA knockdown limits cancer cell proliferation and reduces cancer stemness. We designed and implemented a dual docking protocol to select virtual ACL inhibitors that were scored among the top 10 percentiles by both the Autodock Vina and the Glamdock algorithms. Via this in silico screens of a focused furoic acid library, we discovered four subtypes of furans and benzofurans as novel ACL inhibitors. The hit rate of our in silico protocol was 45.8% with 11 of 24 virtual hits confirmed as active in an in vitro ACL enzymatic assay. The IC₅₀ of the most potent ACL inhibitor A1 is 4.1 μM. Our results demonstrated remarkable hit rate by the dual docking approach and provided novel chemical scaffolds for the development of ACL inhibitors for the treatment of cancer.     

ATP-citrate lyase: a mini-review

Obesity is an increasing world problem that may cause several metabolic complications including insulin resistance, hyperlipidemia, hypertension, and atherosclerosis. Development of therapeutic drugs for obesity has been proven difficult. Current strategies for weight reduction are inhibition of food intake through the central nervous system or blocking the absorption of lipids in the gut. These therapies have many side effects, so new treatments are urgently needed. Fat loss could also be achieved through a decrease in the size and number of adipocytes through apoptosis. Apoptosis is a normal phenomenon of cell death for the purpose of maintaining homeostasis. Induction of apoptosis is a reasonable way to remove adipocytes in obese patients. It is reported that several adipokines and natural products play roles in induction of adipocyte apoptosis. Here we review the recent progress of the roles and mechanisms of adipocyte apoptosis induced by leptin, tumor necrosis factor-α (TNF-α), and natural compounds.     

Fat cell protein phosphorylation. Identification of phosphoprotein-2 as ATP-citrate lyase.

We have purified to apparent homogeneity a phosphoprotein from rat adipose tissue which is rapidly phosphorylated in vitro by ATP. The native phosphoprotein has an approximate sedimentation coefficient of 14.8 S. On sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, the protein dissociated into identical subunits of Mr = 128,000. A phosphoprotein with similar properties was also isolated from liver. Purified phosphoproteins from fat cells and liver had ATP-citrate lyase activity and co-migrated on sodium dodecyl sulfate gels with fat cell phosphoprotein-2, the phosphorylation of which is increased by incubating fat cells with insulin. The phosphoamino acid residue of the cells with insulin. The phosphoamino acid residue of the phosphoprotein was identified as tau-phosphohistidine. These evidences suggest that fat cell phosphoprotein-2 is ATP-citrate lyase.     

ATP-citrate lyase activity in fungal extracts

ATP-citrate lyase activity has been demonstrated in crude extracts from several species ofMortierella. This filamentous fungus characteristically stores lipid in the mycelium. The enzyme catalyses an ATP-dependent cleavage of citrate in the presence of CoA to oxaloacetic acid and acetylCoA. A partially purified preparation has been obtained and shown to have properties similar to preparations isolated from other sources. It is highly specific for citrate and ATP and has an optimum pH value for activity of 8.4. When the fungus is grown in the presence of increasing amounts of glucose the specific activity of the enzyme also increases.     

ATP-citrate lyase deficiency in the mouse

ATP-citrate lyase (Acly) is one of two cytosolic enzymes that synthesize acetyl-coenzyme A (CoA). Because acetyl-CoA is an essential building block for cholesterol and triglycerides, Acly has been considered a therapeutic target for hyperlipidemias and obesity. To define the phenotype of Acly-deficient mice, we created Acly knockout mice in which a beta-galactosidase marker is expressed from Acly regulatory sequences. We also sought to define the cell type-specific expression patterns of Acly to further elucidate the in vivo roles of the enzyme. Homozygous Acly knockout mice died early in development. Heterozygous mice were healthy, fertile, and normolipidemic on both chow and high fat diets, despite expressing half-normal amounts of Acly mRNA and protein. Fibroblasts and hepatocytes from heterozygous Acly mice contained half-normal amounts of Acly mRNA and protein, but this did not perturb triglyceride and cholesterol synthesis or the expression of lipid biosynthetic genes regulated by sterol regulatory element-binding proteins. The expression of acetyl-CoA synthetase 1, another cytosolic enzyme for producing acetyl-CoA, was not up-regulated. As judged by beta-galactosidase staining, Acly was expressed ubiquitously but was expressed particularly highly in tissues with high levels of lipogenesis, such as in the livers of mice fed a high-carbohydrate diet. beta-Galactosidase staining was intense in the developing brain, in keeping with the high levels of de novo lipogenesis of the tissue. In the adult brain, beta-galactosidase staining was in general much lower, consistent with reduced levels of lipogenesis; however, beta-galactosidase expression remained very high in cholinergic neurons, likely reflecting the importance of Acly in generating acetyl-CoA for acetylcholine synthesis. The Acly knockout allele is useful for identifying cell types with a high demand for acetyl-CoA synthesis.     

ATP-citrate lyase as a substrate of protein histidine phosphatase in vertebrates

The first protein histidine phosphatase from vertebrates discovered recently was found in a variety of tissues, however, a physiological substrate protein was missing. Phosphorylation of liver extracts in the presence of EDTA, followed by SDS-PAGE and autoradiography showed labeling of three proteins. Acid- and alkaline-treatment revealed the existence of N-phosphates. Addition of histidine phosphatase exclusively resulted in dephosphorylation of a 110kDa protein (denaturing conditions). Gelfiltration revealed its native molecular mass of approximately 450kDa. That protein was purified and identified as ATP-citrate lyase. The results are in favor of histidine phosphatase playing an important yet unidentified role in metabolic processes.     

Physiological characterization of ATP-citrate lyase in Aspergillus niger

Acetyl-CoA, an important molecule in cellular metabolism, is generated in multiple subcellular compartments and mainly used for energy production, biosynthesis of a diverse set of molecules, and protein acetylation. In eukaryotes, cytosolic acetyl-CoA is derived mainly from the conversion of citrate and CoA by ATP-citrate lyase. Here, we describe the targeted deletions of acl1 and acl2, two tandem divergently transcribed genes encoding subunits of ATP-citrate lyase in Aspergillus niger. We show that loss of acl1 or/and acl2 results in a significant decrease of acetyl-CoA and citric acid levels in these mutants, concomitant with diminished vegetative growth, decreased pigmentation, reduced asexual conidiogenesis, and delayed conidial germination. Exogenous addition of acetate repaired the defects of acl-deficient strains in growth and conidial germination but not pigmentation and conidiogenesis. We demonstrate that both Acl1 and Acl2 subunits are required to form a functional ATP-citrate lyase in A. niger. First, deletion of acl1 or/and acl2 resulted in similar defects in growth and development. Second, enzyme activity assays revealed that loss of either acl1 or acl2 gene resulted in loss of ATP-citrate lyase activity. Third, in vitro enzyme assays using bacterially expressed 6His-tagged Acl protein revealed that only the complex of Acl1 and Acl2 showed ATP-citrate lyase activity, no enzyme activities were detected with the individual protein. Fourth, EGFP-Acl1 and mCherry-Acl2 proteins were co-localized in the cytosol. Thus, acl1 and acl2 coordinately modulate the cytoplasmic acetyl-CoA levels to regulate growth, development, and citric acid synthesis in A. niger.     

ATP-citrate lyase is essential for macrophage inflammatory response

Growing evidence suggests that energy metabolism and inflammation are closely linked and that cross-talk between these processes is fundamental to the pathogenesis of many human diseases. However, the molecular mechanisms underlying these observations are still poorly understood. Here we describe the key role of ATP-citrate lyase (ACLY) in inflammation. We find that ACLY mRNA and protein levels markedly and quickly increase in activated macrophages. Importantly, ACLY activity inhibition as well as ACLY gene silencing lead to reduced nitric oxide, reactive oxygen species and prostaglandin E2 inflammatory mediators. In conclusion, we present a direct role for ACLY in macrophage inflammatory metabolism.     

Synthesis and evaluation of (+) and (-)-2,2-difluorocitrate as inhibitors of rat-liver ATP-citrate lyase and porcine-heart aconitase.

The enantiomers (+) and (-)-2,2-difluorocitrate have been synthesized. Both are good inhibitors of ATP-citrate lyase, showing competitive inhibition against citrate, with Kis = 0.7 microM for (+)-2,2-difluorocitrate and 3.2 microM for (-)-2,2-difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon-carbon bond cleavage as a substrate and there is no evidence of irreversible time-dependent inactivation. When ATP-citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate-induced rate for the (+)-enantiomer and 2% for the (-)-enantiomer. 19F-NMR studies confirm that only the (+)-enantiomer is chemically processed. The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (-)-2,2-Difluorocitrate is a competitive inhibitor against citrate (Kis = 1.5 microM), whereas the (+)-enantiomer is a relatively poor mixed inhibitor (Ki greater than 300 microM). The (-)-enantiomer irreversibly inactivates aconitase at 1.1 min-1.mM-1 at 25 degrees C and pH 7.4, whereas no irreversible inhibition is seen with the (+)-enantiomer. Therefore, it would be expected that the (+)-enantiomer would slow the rate of acetyl-CoA synthesis in vivo, without inhibiting the citric acid cycle.     

Cloning and expression of a human ATP-citrate lyase cDNA.

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.     

Purification and some properties of ATP-citrate lyase from rat brain

ATP-citrate lyase has been purified from rat brain by a new procedure which yields an enzyme of specific activity of 21 U/mg protein (37 °C) (2050-fold purification). Purity (by sodium dodecyl sulfate-gel electrophoresis) of the preparation was comparable to that of rat liver ATP-citrate lyase of similar specific activity. Both brain and liver ATP-citrate lyase have the same electrophoretic mobility, as well as the same immunoreactivity against specific rabbit anti-rat liver ATP-citrate lyase antibody. These data indicate that rat brain ATP-citrate lyase is similar or identical to that present in rat liver. Intraperitoneally injected ³²P_i was incorporated into the structural phosphate of ATP-citrate lyase in rat liver but not into the rat brain enzyme.     

Stoichiometry of phosphorylation of hepatic ATP-citrate lyase by protein kinase

Hepatic ATP-citrate lyase prepared with a fluoride-free step to allow endogenous phosphatases to dephosphorylate the enzyme was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase and [γ-³²P]ATP. After electrophoresis the radioactive phosphate was located predominantly in the gel slice containing the Coomassie blue stained protein corresponding to ATP-citrate lyase. The Stoichiometry of phosphorylation of hepatic ATP-citrate lyase in vitro by the catalytic subunit was such that 0.53 ± 0.02 molecules of phosphate were incorporated per subunit. The degree of phosphorylation was independent of the amount of ATP-citrate lyase present as substrate in the concentration range 1.2–6.4 μm. In the absence of catalytic subunit there was very little labeled phosphate incorporated into ATP-citrate lyase. Phosphorylation of ATP-citrate lyase by catalytic subunit was abolished by the specific protein inhibitor of cyclic AMP-dependent protein kinase. When ATP-citrate lyase was subjected to electrophoresis under nondenaturing conditions, lyase activity was recovered from the gel slice corresponding to the Coomassie blue staining phosphoprotein of a stained gel run in parallel.     

Bromophenols as Candida albicans isocitrate lyase inhibitors

A new series of bromophenols was synthesized by reactions of corresponding phenol analogs with bromine. The synthesized compounds were tested for inhibitory activity against isocitrate lyase (ICL) of Candida albicans and antimicrobial activity against gram-positive and, gram-negative bacteria and fungi. Among the synthesized bromophenols, bis(3-bromo-4,5-dihydroxyphenyl)methanone (11) and (3-bromo-4,5-dihydroxyphenyl)(2,3-dibromo-4,5-dihydroxyphenyl)methanone (12) displayed potent inhibitory activities against ICL, showing a stronger inhibitory effects than were found with natural bromophenol 1. The preliminary structure-activity relationships were investigated in order to determine the essential structural requirements for the inhibitory activities of these compounds against ICL of C. albicans.     

The identification of ATP-citrate lyase as a protein kinase B (Akt) substrate in primary adipocytes

Protein kinase B (Akt) plays a central role in cellular regulation, although many of the physiologically relevant substrates for the kinase remain to be identified. In this study, we have isolated a protein from primary epididymal adipocytes with an apparent molecular weight of 125,000. This protein exhibited immunoreactivity, in an insulin-dependent manner, with a phosphospecific antibody raised against the protein kinase B substrate consensus sequence RXRXX(pS/pT) as well as a phosphospecific antibody that recognizes serine 21/9 of GSK-3alpha/beta. MALDI-TOF mass spectrometry revealed the protein to be ATP-citrate lyase, suggesting that the two phosphospecific antibodies recognize phosphoserine 454, a previously reported insulin- and isoproterenol-stimulated ATP-citrate lyase phosphorylation site. Indeed, both insulin and isoproterenol stimulated the phosphorylation of this protein on the site recognized by the phosphospecific antibodies in a wortmannin-sensitive and -insensitive manner, respectively. In addition, transient expression of a constitutively active protein kinase B in primary adipocytes mimicked the effect of insulin on ATP-citrate lyase phosphorylation. Furthermore, ATP-citrate lyase was phosphorylated in vitro by recombinant protein kinase B on the same site. Taken together, these results demonstrate that serine 454 of ATP-citrate lyase is a novel and major in vivo substrate for protein kinase B.     

Inhibition of ATP-citrate lyase improves NASH,liver fibrosis,and dyslipidemia

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Both subunits of ATP-citrate lyase from Chlorobium tepidum contribute to catalytic activity

ATP-citrate lyase (ACL) is an essential enzyme of the reductive tricarboxylic acid (RTCA) pathway of CO(2) assimilation. The RTCA pathway occurs in several groups of autotrophic prokaryotes, including the green sulfur bacteria. ACL catalyzes the coenzyme A (CoA)-dependent and MgATP-dependent cleavage of citrate into oxaloacetate and acetyl-CoA, representing a key step in the RTCA pathway. To characterize this enzyme from the green sulfur bacterium Chlorobium tepidum and determine the role of its two distinct polypeptide chains, recombinant holo-ACL as well as its two individual subunit polypeptides were synthesized in Escherichia coli. The recombinant holoenzyme, prepared from coexpressed large and small ACL genes, and the individual large and small subunit polypeptides, prepared from singly expressed genes, were all purified to homogeneity to high yield. Purified recombinant holo-ACL was isolated at high specific activity, and its k(cat) was comparable to that of previously prepared native C. tepidum ACL. Moreover, the purified recombinant large and small subunit polypeptides were able to reconstitute the holo-ACL in vitro, with activity levels approaching that of recombinant holo-ACL prepared from coexpressed genes. Stoichiometric amounts of each subunit protein were required to maximize the activity and form the most stable structure of reconstituted holo-ACL. These results suggested that this reconstitution system could be used to discern the catalytic role of specific amino acid residues on each subunit. Reconstitution and mutagenesis studies together indicated that residues of each subunit contributed to different aspects of the catalytic mechanism, suggesting that both subunit proteins contribute to the active site of C. tepidum ACL.