Human ACC2 Research Articles

Filament structures unveil the dynamic organization of human acetyl-CoA carboxylase.

Human acetyl-coenzyme A (CoA) carboxylases (ACCs) catalyze the carboxylation of acetyl-CoA, which is the rate-limiting step in fatty acid synthesis. The molecular mechanism underlying the dynamic organization of ACCs is largely unknown. Here, we determined the cryo-electron microscopy (EM) structure of human ACC1 in its inactive state, which forms a unique filament structure and is in complex with acetyl-CoA. We also determined the cryo-EM structure of human ACC1 activated by dephosphorylation and citrate treatment, at a resolution of 2.55 Å. Notably, the covalently linked biotin binds to a site that is distant from the acetyl-CoA binding site when acetyl-CoA is absent, suggesting a potential coordination between biotin binding and acetyl-CoA binding. These findings provide insights into the structural dynamics and regulatory mechanisms of human ACCs.

CircACC1 Regulates Assembly and Activation of AMPK Complex under Metabolic Stress

We report that circACC1, a circular RNA derived from human ACC1, plays a critical role in cellular responses to metabolic stress. CircACC1 is preferentially produced over ACC1 in response to serum deprivation by the transcription factor c-Jun. It functions to stabilize and promote the enzymatic activity of the AMPK holoenzyme by forming a ternary complex with the regulatory β and γ subunits. The cellular levels of circACC1 modulate both fatty acid β-oxidation and glycolysis, resulting in profound changes in cellular lipid storage. In a tumor xenograft model, silencing or enforced expression of circACC1 resulted in growth inhibition and enhancement, respectively. Moreover, increased AMPK activation in colorectal cancer tissues was frequently associated with elevated circACC1 expression. We conclude that circACC1 serves as an economic means to elicit AMPK activation and moreover propose that cancer cells exploit circACC1 during metabolic reprogramming.

Structural Characterization of i-Motif Structure in the Human Acetyl-CoA Carboxylase 1 Gene Promoters and Their Role in the Regulation of Gene Expression.

The polypurine/polypyrimidine-rich sequences within the promoters (PI and PII) of human acetyl coenzyme A (CoA) carboxylase 1 (ACC1) gene play a vital role in determining hormone- or diet-inducible expression of ACC1. PI and PII contain consecutive runs of three and three to five G/C base pairs, respectively. In a previous study, G-rich DNA sequences of human ACC1 PI and PII were found to fold into G-quadruplex structures; these consequently acted as strong barriers to transcription and DNA replication. Typically, stretches of C-rich sequences that coexist with stretches of guanines have the capacity to form another four-stranded secondary structure known as an i-motif. However, studies on the i-motif structure are limited and its functional significance is unclear. In the current study, through the use of a combination of different techniques, it is demonstrated that C-rich single-stranded DNA derived from ACC1 PI and PII form intramolecular i-motif structures and affect normal DNA metabolic processes. Additionally, the C-rich strands of PI and PII in duplex DNA adopt the i-motif conformation in crowded solution environments at neutral pH. Notably, the i-motif-forming sequences of PI and PII suppressed luciferase gene transcription in HeLa cells. Furthermore, substitution of a nucleotide sequence that has no potential to form the i-motif structure increases luciferase gene expression in HeLa cells. These results support the idea that C-rich sequences within ACC1 PI and PII can form intramolecular i-motif structures, cause suppression of transcription, and thus reveal the functional significance of C-rich sequences in the regulation of ACC1 gene expression.

Combined 3D-QSAR modeling and molecular docking study on spiro-derivatives as inhibitors of acetyl-CoA carboxylase

Acetyl-CoA carboxylases (ACC) have long been recognized as an attractive target for metabolic syndrome because of its indispensable role in the regulation of fatty acid metabolism in most living organisms. Based on the lead compound CP-640186, a series of spiro-compounds of benzothiazole derivatives have been identified as ACC inhibitors, which displayed high ACC inhibitor activities and good metabolic stability. Nevertheless, no quantitative structure-activity relationship study has been developed for this kind of ACC inhibitors. In order to evaluate the correlation between changes on structures and variation on biological activity, three-dimensional quantitative structure-activity relationship analysis was employed. Satisfactory results were obtained after performing a leave-one-out cross-validation study with q 2 and r 2 values of 0.625 and 0.927 by the comparative molecular field analysis model, 0.779 and 0.937 by the comparative molecular similarity indices analysis model, respectively. The predictive ability of the models was validated using external validation test set. The contour map analysis of comparative molecular field analysis and comparative molecular similarity indices analysis models indicated that electropositive and bulky groups substituted at the R 1 position of carbamido group, and hydrophilic groups substituted at the phenyl ring were favorable for the inhibitory activity. Furthermore, FlexX docking was employed to investigate the binding mode between human ACC1 and its inhibitors and to validate the three-dimensional quantitative structure-activity relationship models. As a result, based on the results of three-dimensional quantitative structure-activity relationship and FlexX docking, a series of ACC inhibitors with higher predictive activities have been designed. This work might provide valuable information in further optimization of human ACC inhibitors.

Abstract 1129: How to develop ACC1 inhibitors targeting lipid metabolism and oncogenic signaling pathways effectively and safely

Abstract Although targeting lipogenesis for cancer treatment appears to have a strong rationale, drug discovery in this field has not been fully explored due to the lack of understanding the mode of action as well as the therapeutic window. We reported previously on a class of novel ACC inhibitors with potent and selective activity against human ACC1, an isoform overexpressed in many cancer types. These ACC inhibitors revealed strong anti-tumor activity, including induction of tumor cell apoptosis in vitro and tumor regression in vivo in a sub-set of tumor models. To further demonstrate the therapeutic potential of ACC inhibitors, we conducted a series of studies in xenograft mice and rat to evaluate the anti-tumor efficacy of ACC inhibitors and to characterize their safety profile. We report that breast, prostate, and pancreatic cancers are among the most sensitive tumors to ACC inhibition. Interestingly, the anti-tumor kinetics correlated with reduction in palmitate levels without substantial changes in structural lipid components. In addition, a sub-type of KRAS mutation and activation of the Wnt pathway correlates with the sensitivity of tumors to ACC inhibitors. Treatment with ACC inhibitors at high doses caused an immediate decrease in food intake and followed with body weight loss. A clear correlation between the reduction of food intake and exposure of ACC inhibitor was observed. Upon withdrawing drug, the effect on food intake is restored. Therefore, we investigated intermittent dosing schedules and food effects on the tolerability and anti-tumor efficacy of ACC inhibitors. We could demonstrate that the tolerability was improved without compromising the efficacy compared to continuous treatment. Furthermore, feeding animals a high fat diet prevented body weight loss and meanwhile maintained the antitumor activity. These results indicate that strong reduction of food intake seems the cause of intolerability, which can be prevented and reversed either by intermittent dosing, or by exogenously supplementing with a high fat diet. Furthermore, for the first time we provided in vivo evidence that exogenous lipids could complement de novo lipogenesis inhibition in normal cells, while tumor growth requires lipogenesis irrespective of existing circulating lipids. In summary, these assessments provide scientific insights and strategy on how to best target tumor lipid metabolism and lipid signaling effectively and safely for cancer therapy. Citation Format: Ningshu Liu, Wilhelm Bone, Sendhil S. Velan, Krishnarao Doddapuneni, Jadegoud Yaligar, Kai Thede Thede, Ursula Moenning, Xiaohe Shi, Xianfeng Tian, Elissaveta Petrova1, Franz von Nussbaum, Dominik Mumberg, Michael Brands, Karl Ziegelbauer. How to develop ACC1 inhibitors targeting lipid metabolism and oncogenic signaling pathways effectively and safely. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1129. doi:10.1158/1538-7445.AM2015-1129

Abstract 2242: Inhibition of acetyl-CoA carboxylase attenuates lipidation and activity of oncogenic signaling pathways in pancreatic cancer

Abstract Normal cells can use circulating lipids, while some tumor cells require de novo biogenesis of fatty acids for survival irrespective of existing exogenous lipids. Acetyl-CoA Carboxylase (ACC) is the rate-limiting enzyme in the process of de novo fatty acid synthesis and one of its isoforms (ACC1) is overexpressed in various cancer types. We have developed a class of novel, potent small molecule inhibitors of ACC, which preferentially inhibit human ACC1 activity. Interestingly, in vitro the compounds are active in a selective subset of cancer cells, regardless of their proliferation rate, and pancreatic cancer cells are among the most sensitive cell lines to ACC1 inhibition both in vitro and in vivo. Notably, pancreatic cancer is characterized by alterations in several oncogenic pathways: KRAS, Hedgehog (Hh) and Wnt. One mechanism that modulates the activity of the above-mentioned oncogenic signaling molecules is protein lipidation. We hypothesized that in pancreatic cancer cells de novo fatty acid synthesis is an important source of lipids necessary for fully acting KRAS, Hh and Wnt molecules, and therefore the process is essential for cell proliferation and survival. To investigate this as potential novel anti-tumor mechanism of action, we conducted a series of experiments to assess the effect of ACC inhibition on these oncogenic pathways in vitro and in vivo. In vitro we first investigated the effect of ACC inhibition on Wnt secretion and activity, a process requiring lipidation of Wnt. Treatment of a Wnt3A-overexpressing cell line, L-Wnt3A, with the ACC inhibitor, indeed, leads to reduced Wnt3A protein levels secreted in the media. This result was further confirmed by the Super8xTOPFlash reporter assay in HEK293 cells incubated with conditional media from L-Wnt3A cells, with or without treatment with the ACC inhibitor. Furthermore, we showed that in Capan-2 cells, in which ligand-dependent autocrine Wnt signaling is active, treatment with the ACC inhibitor decreased the expression of β-catenin target genes and cell proliferation. In vivo profiling of the ACC inhibitor in a set of patient derived xenograft (PDX) models of pancreatic cancer indicated a correlation between the sensitivity to ACC inhibition and aberrant activation of Hh and Wnt signaling in the tumors, as shown by immunohistochemical detection of downstream targets of the two pathways. Currently, we are investigating the specific changes in Hh and Wnt signaling, following ACC inhibition in both a PDX model of pancreatic cancer, as well as in a Capan-2 xenograft mouse model. Together, these studies are building strong support for the hypothesis that tumor sensitivity to de novo fatty acid synthesis inhibition is linked to the dependence on multiple oncogenic signaling pathways that are regulated by protein lipidation, and highlight a potential new general approach for targeting ACC for the treatment of cancers. Citation Format: Elissaveta Petrova, Arne Scholz, Andrea Sturz, Juliane Paul, Franziska Siegel, Dominik Mumberg, Ningshu Liu. Inhibition of acetyl-CoA carboxylase attenuates lipidation and activity of oncogenic signaling pathways in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2242. doi:10.1158/1538-7445.AM2015-2242

Abstract 5460: Intracellular uptake and metabolism of SH7139: Is it a targeted prodrug for B-cell lymphomas

Abstract Selective high affinity ligands (SHALs) are novel targeting agents composed of lysine/mini-polyethylene glycol scaffolds linking two or three ‘recognition’ ligands together. The tridentate SHAL SH7139 is selectively cytotoxic at sub-nanomolar concentrations to lymphoma cells over-expressing HLA-DR10. Electron microscopy examinations of Burkitt's lymphoma xenografts from mice treated with SH7139 suggest one mechanism for cell killing may involve cell signaling-induced apoptosis analogous to that observed with Lym-1 antibody. An alternative mechanism is that intracellular hydrolysis of the SHAL's amide bonds following its uptake and metabolism by lymphoma cells could lead to the release of the three recognition ligands and the subsequent inhibition (or promotion) of cellular activities that trigger apoptosis or induce cell death and necrosis. Each of the three recognition ligands (Dv, Ct, and Cb) is independently, but non-selectively, toxic to both Raji (HLA-DR10 positive) and Jurkat (HLA-DR10 negative) cells. The Ct ligand, 3-(2-([3-chloro-5-trifluoromethyl)-2-pyridinyl]oxy)-anilino)-3-oxopropanionic acid, is a structural analog of aryloxyphenoxypropionate herbicides that inhibit acetyl-CoA carboxylase (ACC), a key enzyme in fatty acid biosynthesis that is upregulated in many cancers to meet their need for rapid growth and proliferation. Our studies show that although Ct could be accommodated into the herbicide binding pocket of ACC, this ligand was not found to inhibit human and rat ACC1 or ACC2. However, metabolic cleavage of the single amide bond present in Ct would lead to the production of two smaller metabolites, an aniline derivative and malonate, both of which are known to be biologically active and potentially toxic compounds. The Dv ligand, dabsyl-L-valine, belongs to a family of biphenylsulfonamides, many of which have been shown to inhibit matrix metalloproteinases such as MMP9 and MMP14 that are upregulated in tumor cells. The Cb ligand, 4-[4-(4-chlorobenzyl)piperazino]-3-nitrobenzene carboxylic acid, is a structural analog of a large number of inhibitors that have been shown to block the assembly of the contractile ring and the initiation of cytokinesis by inhibiting guanosine nucleoside binding to MgcRacGAP, a protein that participates in the activation of the myosin motor and the initiation of cleavage furrow ingression. A combination of SH7139 metabolism studies, computer modeling, and enzyme inhibition assays have been performed to determine if Ct, Dv and Cb and their metabolites are produced and whether they contribute to lymphoma cell killing. Citation Format: Monique C. Balhorn, Gary Mirick, Dong Cheng, Zhengping Ma, Edmond Y. Lau, Saphon Hok, Gerald L. DeNardo, Rod Balhorn. Intracellular uptake and metabolism of SH7139: Is it a targeted prodrug for B-cell lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5460. doi:10.1158/1538-7445.AM2014-5460

Abstract 2679: Acetyl-CoA carboxylase inhibition by ND646 reduces fatty acid synthesis and inhibits cell proliferation in human non-small cell lung cancer cells

Abstract Continuous de novo lipogenesis is a common feature of tumor cells and is required to meet the bioenergetic and biosynthetic demands of a growing tumor. The expression and activity of the fatty acid synthetic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase are up-regulated in many types of cancer, where lipogenesis is essential for proliferation and tumor cell survival. These enzymes are therefore attractive targets for anti-neoplastic intervention. Inhibition of ACC results in inhibition of fatty acid synthesis (FASyn) and stimulation of fatty acid oxidation in cultured cells and in animals and has been shown to reduce cancer cell growth in vitro. Our efforts to develop novel ACC inhibitors have focused on the subunit dimerization site of the biotin carboxylase (BC) domain of the enzyme. This site also binds the inhibitory phosphopeptide of ACC phosphorylated by AMPK and the fungal metabolite Soraphen A, both of which suppress dimerization and inhibit enzymatic activity. Using state-of-the-art structure-based drug design and crystal structures of human ACC2 BC domain, we have identified a unique series of allosteric inhibitors with low nanomolar potency, represented by ND646, that bind to the Soraphen binding site, exhibit potent and selective activity in vitro and in vivo and show anti-neoplastic properties in cultured human non-small cell lung cancer (NSCLC) cells. The anti-proliferative action of ND646 in NSCLC cell lines A549, H460, and H358 was enhanced in delipidated media and attenuated by supplementation with palmitic acid, suggesting that the anti-proliferative effect of ND646 is induced by depletion of cellular fatty acids. Current efforts are focused on determining if the cell growth inhibitory actions of ND646 also possess apoptotic properties and on assessing the anti-neoplastic activity of ND646 in preclinical models of lung cancer. During the course of these studies, we also discovered that interaction of ND646 with the ACC phosphopeptide binding site markedly reduced the phosphorylation state of the enzyme in A549 cells, as determined by western blot analysis using anti-phosphoACC (pACC) antibody. Furthermore, in HepG2 cells, the reduction in pACC by the related analog ND630 (EC50 = 66 nM), which closely paralleled inhibition of FASyn (EC50 = 42 nM), resulted in complete loss of pACC at high doses. These observations, that were corroborated in hepatic tissue isolated from both ND646 and ND630 treated mice and in situ in tumors of genetically engineered mouse models of lung cancer dosed with ND646, suggest that assessment of pACC could provide a sensitive biomarker of target engagement for ACC inhibitors interacting in this region of the enzyme. Additional efforts are focused on examining the genetic contexts and optimal therapeutic combinations such that ACC inhibitors may find the greatest clinical utility as anti-neoplastic agents. Citation Format: Robert Svensson, Geraldine Harriman, Jeremy Greenwood, Sathesh Bhat, H.James Harwood, Rosana Kapeller, Reuben Shaw. Acetyl-CoA carboxylase inhibition by ND646 reduces fatty acid synthesis and inhibits cell proliferation in human non-small cell lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2679. doi:10.1158/1538-7445.AM2014-2679

Abstract 1427: Liver selective Acetyl-CoA Carboxylase inhibition by ND-654 decreases hepatocellular carcinoma development in cirrhotic rats

Abstract Background: Hepatocellular carcinoma (HCC) is increasing in incidence worldwide. This increase is in part due to the obesity epidemic which is an independent risk factor for both nonalcoholic fatty liver disease (NAFLD) and HCC. In fact, recent evidence suggests that enhanced de novo lipogenesis is a critical mediator for HCC development. Current treatment options for HCC are limited, and as such, prognosis is extremely poor with a 5-year survival less than 12%. Therefore, there is an urgent medical need for new therapeutic strategies. Simultaneous inhibition of the acetyl-CoA carboxylase isozymes, ACC1 and ACC2, results in concomitant inhibition of fatty acid synthesis (FASyn) and stimulation of fatty acid oxidation (FAOxn) in cultured cells and in animals, reduces hepatic steatosis, and impedes tumor cell growth in vitro. Here, we test the hypothesis that simultaneous reduction of hepatic FASyn through inhibition of ACC1 and stimulation of hepatic FAOxn through inhibition ACC2 is a potential therapeutic target for HCC. Results: Using state-of-the-art structure-based drug design and crystal structures of the human ACC2 biotin carboxylase domain, we identified ND-654, a hepatoselective (2700:5:1 liver to plasma to muscle exposure), allosteric inhibitor that binds to the ACC subunit dimerization site, inhibits the enzymatic activity of both ACC1 (IC50 = 3 nM) and ACC2 (IC50 = 8 nM), and inhibits FASyn in HepG2 cells (IC50 = 14 nM) and in rats (ED50 = 0.3 mg/kg). Male Wistar rats were treated weekly with 50 mg/kg diethylnitrosamine (DEN) to induce sequential development of fibrosis, cirrhosis and HCC. After establishment of cirrhosis (13 wks), rats were treated daily by oral gavage with either vehicle or 10 mg/kg ND-654. At the end of the study (18 wks), rats were sacrificed and tumor nodules were counted. Liver tissue and plasma were also analyzed to assess the effects of ND-654 on disease progression. When measured four hours after the final dose, ND-654 showed preferential liver exposure, with steady state levels reaching 3.7±1.8 µM in cirrhotic tissue and 2.2±1.8 µM in tumor tissue. ND-654 did not alter body weight or food consumption, but reduced serum triglyceride levels by 35%. Importantly, ND-654 significantly (p < 0.01) inhibited HCC development in DEN-injured livers from an average of 15.8 tumor nodules in controls to an average of 7.1 tumor nodules in ND-654-treated rats. Consistently, ND-654 reduced the DEN-induced increase in liver weight by 48%, and decreased the number of tumor related deaths by 67% (6 of 8 controls vs 2 of 8 ND-654-treated rats). Conclusions: These results provide further evidence that de novo lipogenesis is an important mediator of hepatic carcinogenesis and that selective inhibition of hepatic ACC is a potential therapeutic strategy for HCC. Citation Format: Danielle K. DePeralta, Lan Wei, Geraldine Harriman, Jeremy Greenwood, Sathesh Bhat, William Westlin, H. James Harwood, Rosana Kapeller, Kenneth K. Tanabe, Bryan C. Fuchs. Liver selective Acetyl-CoA Carboxylase inhibition by ND-654 decreases hepatocellular carcinoma development in cirrhotic rats. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1427. doi:10.1158/1538-7445.AM2014-1427

Human acetyl-CoA carboxylase 2 expressed in silkworm Bombyx mori exhibits posttranslational biotinylation and phosphorylation.

Biotin-dependent human acetyl-CoA carboxylases (ACCs) are integral in homeostatic lipid metabolism. By securing posttranslational biotinylation, ACCs perform coordinated catalytic functions allosterically regulated by phosphorylation/dephosphorylation and citrate. The production of authentic recombinant ACCs is heeded to provide a reliable tool for molecular studies and drug discovery. Here, we examined whether the human ACC2 (hACC2), an isoform of ACC produced using the silkworm BmNPV bacmid system, is equipped with proper posttranslational modifications to carry out catalytic functions as the silkworm harbors an inherent posttranslational modification machinery. Purified hACC2 possessed genuine biotinylation capacity probed by biotin-specific streptavidin and biotin antibodies. In addition, phosphorylated hACC2 displayed limited catalytic activity whereas dephosphorylated hACC2 revealed an enhanced enzymatic activity. Moreover, hACC2 polymerization, analyzed by native page gel analysis and atomic force microscopy imaging, was allosterically regulated by citrate and the phosphorylation/dephosphorylation modulated citrate-induced hACC2 polymerization process. Thus, the silkworm BmNPV bacmid system provides a reliable eukaryotic protein production platform for structural and functional analysis and therapeutic drug discovery applications implementing suitable posttranslational biotinylation and phosphorylation.

Expression of Fat and Cholesterol Biomarkers in Meat Goats

Consumption of high amounts of saturated fatty acids in meat has been implicated in the onset of cardiovascular disease. Chevon (goat meat) is higher in mono-unsaturated and poly-unsaturated fatty acids than beef and lamb. Limited data is available on the expression of fat and cholesterol biomarkers in meat goats. The objective of this experiment was to determine expression of Acetyl-CoA Carboxylase (ACC1), Apoplipoproteins, A (ApoA1), and B (ApoB) in different breeds of meat goats. Protein sequence alignments were generated to determine conservation for antibody selection. The motif (SMS79pGL) was conserved in the goat, human, mouse, rat and bovine ACC1 proteins. The ApoA1 and ApoB protein alignments (human, bovine and rabbit) revealed high protein sequence homology. The Enzyme-Linked Immunosorbent Assay (ELISA) was used to determine serum ACC1, ApoA1 and ApoB in Spanish and Myotonic goats. Spanish goats had higher (P<0.05) ACC1 than Myotonic goats. There was a gender effect (P<0.05) where females expressed more ACC1 than males. Breed and gender differences were detected in Spanish and Myotonic goats, with Spanish goats showing 37% more (P<0.05) ApoA1 expression in the blood than Myotonic goats and female goats with 47% higher expression of ApoA1 than males. Inversely, Myotonic goats expressed 35% higher (P<0.05) levels of ApoB than Spanish goats and males had 46% higher (P<0.05) ApoBexpression than females. These data demonstrate that inherent differences exist in lipid metabolism of meat goats and can lead to lipid biomarker assisted breeding programs to produce a heart, healthy red meat for human consumption.

Abstract 4617: BAY-ACC001, a novel ACC inhibitor, regulates fatty acids synthesis and lipid survival signaling with promising in vitro and in vivo activities in multiple preclinical tumor models.

Abstract Unlike normal cells, tumors undergo intensified de novo biogenesis of fatty acids (FAs) irrespective of the available circulating lipids. Acetyl-CoA carboxylase 1 (ACC) controls the rate limiting step in FA synthesis. ACC1 is up-regulated in a variety of human tumors and strongly associated with poorer prognosis in some tumor indications. Although targeting lipogenesis for cancer treatment appeared having strong rationale, drug discovery in this field has not been fully explored due to the lack of powerful tools for both evaluation and understanding the mode of action. Here we report the identification and the functional characterization of a highly selective ACC inhibitor BAY-ACC001, a ketoenol derivative using various pharmacological and lipidomic approaches to address the mechanism of ACC inhibition in cancer cells and its efficacy in preclinical tumor models. BAY-ACC001 inhibits human ACC1 and ACC1 with biochemical IC50s of 278 nM and 2590 nM measured by ACC1- or ACC2- mediated generation of ADP, respectively. In a cellular mechanistic assay using MCF7 tumor cells, BAY-ACC001 potently inhibited malonyl-CoA synthesis with an IC50 of 62 nM. Profiling BAY-ACC001 in a panel of 100 tumor cell lines revealed strong anti-proliferative activity in a sub-set of tumor cell lines with IC50s at low 3-digit nanomolar. Of note, in contrast to the potent activity in tumor cell lines, e.g. apoptosis induction in MCF7 breast tumor cells, ACC inhibitor showed only weak anti-proliferative effect and could not induce apoptosis in a set of non-transformed mammary epithelial cells. To elucidate the anti-tumor MoA, the levels of lipid components (∼400 lipid molecules) in MCF7 cells were analyzed using lipidomic technology. Interestingly, ACC inhibition did not lead to a simple depletion of lipid in cells, evident from a significant increase in tumor apoptosis-related lipid signaling molecules ceramides. These results suggested a bi-direction linkage between FA synthesis and the regulation of tumor cell survival. Single administration of BAY-ACC001 orally at 10 mg/kg (the maximum tolerable dose is 30 mg/kg, BID) in mice strongly reduced malonyl CoA levels in tumors. Treatment of BAY-ACC001 as single agent was efficacious in multiple tumor models, including MCF7 (breast), PC3 (prostate), HCT116 (colon) and MDA-MB-435 (melanoma) xenograft models; partial tumor remissions observed in MCF7 model. Furthermore, combination with Tamoxifen was synergistic in the MCF7 tumor model in vitro and in vivo. In conclusion, the prevalence of the exacerbated de novo FA synthesis observed in primary and metastatic tumors, the ACC-mediated novel survival signal transduction discovered in this study, and robust in vitro and in vivo anti-tumor activity of BAY-ACC001 provide a strong rationale for developing novel therapeutics targeting ACC for cancer treatment. Citation Format: Ningshu Liu, Maher Najar, Arne Scholz, Knut Eis, Ulf Bömer, Philip Lienau, Kai Thede, Dominik Mumberg, Michael Brands, Karl Ziegelbauer. BAY-ACC001, a novel ACC inhibitor, regulates fatty acids synthesis and lipid survival signaling with promising in vitro and in vivo activities in multiple preclinical tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4617. doi:10.1158/1538-7445.AM2013-4617

Inhibition of acetyl-CoA carboxylase 2 enhances skeletal muscle fatty acid oxidation and improves whole-body glucose homeostasis in db/db mice

Excessive ectopic lipid deposition contributes to impaired insulin action in peripheral tissues and is considered an important link between obesity and type 2 diabetes mellitus. Acetyl-CoA carboxylase 2 (ACC2) is a key regulatory enzyme controlling skeletal muscle mitochondrial fatty acid oxidation; inhibition of ACC2 results in enhanced oxidation of lipids. Several mouse models lacking functional ACC2 have been reported in the literature. However, the phenotypes of the different models are inconclusive with respect to glucose homeostasis and protection from diet-induced obesity. Here, we studied the effects of pharmacological inhibition of ACC2 using as a selective inhibitor the S enantiomer of compound 9c ([S]-9c). Selectivity was confirmed in biochemical assays using purified human ACC1 and ACC2. (S)-9c significantly increased fatty acid oxidation in isolated extensor digitorum longus muscle from different mouse models (EC(50) 226 nmol/l). Accordingly, short-term treatment of mice with (S)-9c decreased malonyl-CoA levels in skeletal muscle and concomitantly reduced intramyocellular lipid levels. Treatment of db/db mice for 70 days with (S)-9c (10 and 30 mg/kg, by oral gavage) resulted in improved oral glucose tolerance (AUC -36%, p < 0.05), enhanced skeletal muscle 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) uptake, as well as lowered prandial glucose (-31%, p < 0.01) and HbA(1c) (-0.7%, p < 0.05). Body weight, liver triacylglycerol, plasma insulin and pancreatic insulin content were unaffected by the treatment. In conclusion, the ACC2-selective inhibitor (S)-9c revealed glucose-lowering effects in a mouse model of diabetes mellitus.

Flavokawain B induces apoptosis of human oral adenoid cystic cancer ACC-2 cells via up-regulation of Bim and down-regulation of Bcl-2 expression

Novel effective drugs are still urgently needed in the prevention and treatment of oral adenoid cystic carcinoma (ACC). In this study, we have assessed the antitumor potential and molecular mechanisms of flavokawain B (FKB) as a kava chalcone on the ACC-2 cell line in vitro. The results demonstrated that FKB could significantly inhibit the cell proliferation of ACC-2 in a dose-dependent manner that was associated with induced apoptosis and cell cycle G2-M arrest, and the half maximal inhibitory concentration (IC50) of flavokawain-B treatment for 48h was estimated to be 4.69± 0.43µmol/L. Mechanistically, FKB could induce the release of cytochrome c from mitochondria into the cytosol, and activate the cleavage of caspase-3 and, eventually, the poly(ADP-ribose) polymerase (PARP), in a dose-dependent manner, leading to marked apoptotic effect of ACC-2 cells. The apoptotic action of FKB was associated with the increased expression of proapoptotic proteins: Bim, Bax, Bak and a decreased expression of antiapoptotic Bcl-2. Among them, Bim expression was significantly induced by FKB, and knockdown of Bim expression by short-hairpin RNAs attenuated the inhibitory effect induced by FKB on ACC-2 cells. These results suggest Bim may be one of the potential transcriptional targets, and suggests the potential usefulness of FKB for the prevention and treatment of ACC.

Structure-guided inhibitor design for human acetyl-coenzyme A carboxylase by interspecies active site conversion.

Inhibition of acetyl-CoA carboxylases (ACCs), a crucial enzyme for fatty acid metabolism, has been shown to promote fatty acid oxidation and reduce body fat in animal models. Therefore, ACCs are attractive targets for structure-based inhibitor design, particularly the carboxyltransferase (CT) domain, which is the primary site for inhibitor interaction. We have cloned, expressed, and purified the CT domain of human ACC2 using baculovirus-mediated insect cell expression system. However, attempts to crystallize the human ACC2 CT domain have not been successful in our hands. Hence, we have been using the available crystal structure of yeast CT domain to design human ACC inhibitors. Unfortunately, as the selectivity of the lead series has increased against the full-length human enzyme, the potency against the yeast enzyme has decreased significantly. This loss of potency against the yeast enzyme correlated with a complete lack of binding of the human-specific compounds to crystals of the yeast CT domain. Here, we address this problem by converting nine key active site residues of the yeast CT domain to the corresponding human residues. The resulting humanized yeast ACC-CT (yCT-H9) protein exhibits biochemical and biophysical properties closer to the human CT domain and binding to human specific compounds. We report high resolution crystal structures of yCT-H9 complexed with inhibitors that show a preference for the human CT domain. These structures offer insights that explain the species selectivity of ACC inhibitors and may guide future drug design programs.

Effects of 125 I seeds interstitial brachytherapy on orbital adenoid cystic carcinoma

Background Adenoid cystic carcinoma is a malignant tumor originating from lacrimal gland epithelium with high recurrence rate and mortality because of its invasiveness. Although surgery, radiotherapy and chemotherapy are used clinically,the curative effect is not enough satisfied. Objective This study was to provide an experimental basis for clinical application of 125 Ⅰ seeds interstitial brachytherapy. Methods Human ACC-2 cell links were transplanted subcutaneously in the back of 30 SPF BALB/C nude mice to establish the ACC model,and 25 of these mice with suitable sizes of tumor were selected and randomly divided into the G1 (0. 4 mCi 125 Ⅰ ), G2 (0. 6 mCi 125 Ⅰ ) ,G3(0. 8mCi 125 Ⅰ ) ,and G4( 1.0mCi 125 Ⅰ ) groups according to the different therapeutic radioactivity treatments,with 5 nude mice for each group,and a 125 Ⅰ seed without radioactivity was used in 5 mice as the control group. The dimensions of the tumors were measured at 2-day intervals and the inhibition rates of tumors were calculated. Nude mice were killed 14 days later by the broken neck method,and the amount of apoptosis and necrosis as well as the maximum effective radius of tumor to 125Ⅰ seed were detected under the transmission electron microscope and routine pathological examination. Results Fourteen day after operation, the dimension of tumors was (3713.19±243.23)mm3 in the G0 group;while in the G1 ,G2 ,G3 and G4 groups,the dimensions of tumors were (3113.35±316.54) mm3,(2635.85±261.21) mm3, (2538.37±312.16) mm3 and(1686.28±231.65) mm3,respectively, showing a significant decrease in comparison with the Go group( P＜0. 05 ). The tumor inhibitory rates in the G1 ,G2,G3 and G4 group were(20. 11±3.09)％, (36. 18±2.54)％ ,(40. 83±4. 17)％ ,and(66. 63±5.34)％ ,with an obvious elevation with the increase in the dose of 125 Ⅰ ( F=120. 240,P=0. 000). Correlative analysis showed that the intensity of radioactivity from 125Ⅰ had a positive correlation with tumor inhibitory rate (r =0. 653,P =0. 008 ). The maximum effective radius were ( 5.2 ±0.5 ) mm, ( 6.4 ±0. 7 ) mm, ( 7.4 ±0.4 ) mm, and ( 8.2 ±0. 5 ) mm in the G1 ,G2, G3 and G4 groups, with the considerable differences among them (F=29. 22, P=0. 000). Radioactivity of 125 Ⅰ exhibited positive correlation with the maximum effective radius ( r =0. 609, P =0. 004). Conclusions 125 Ⅰseed implantation brachytherapy can inhibit the growth of the transplanted ACC in BALB/C nude mice by suppressing the proliferation of tumor cells. It is a safe ,feasible and effective method to treat adenoid cystic carcinoma. Key words: 125 Ⅰ seeds; Adenoid cystic carcinoma; Brachytherapy

Design of small molecule inhibitors of acetyl-CoA carboxylase 1 and 2 showing reduction of hepatic malonyl-CoA levels in vivo in obese Zucker rats

Inhibition of acetyl-CoA carboxylases has the potential for modulating long chain fatty acid biosynthesis and mitochondrial fatty acid oxidation. Hybridization of weak inhibitors of ACC2 provided a novel, moderately potent but lipophilic series. Optimization led to compounds 33 and 37, which exhibit potent inhibition of human ACC2, 10-fold selectivity over inhibition of human ACC1, good physical and in vitro ADME properties and good bioavailability. X-ray crystallography has shown this series binding in the CT-domain of ACC2 and revealed two key hydrogen bonding interactions. Both 33 and 37 lower levels of hepatic malonyl-CoA in vivo in obese Zucker rats.

Recombinant yeast screen for new inhibitors of human acetyl-CoA carboxylase 2 identifies potential drugs to treat obesity

Acetyl-CoA carboxylase (ACC) is a key enzyme of fatty acid metabolism with multiple isozymes often expressed in different eukaryotic cellular compartments. ACC-made malonyl-CoA serves as a precursor for fatty acids; it also regulates fatty acid oxidation and feeding behavior in animals. ACC provides an important target for new drugs to treat human diseases. We have developed an inexpensive nonradioactive high-throughput screening system to identify new ACC inhibitors. The screen uses yeast gene-replacement strains depending for growth on cloned human ACC1 and ACC2. In "proof of concept" experiments, growth of such strains was inhibited by compounds known to target human ACCs. The screen is sensitive and robust. Medium-size chemical libraries yielded new specific inhibitors of human ACC2. The target of the best of these inhibitors was confirmed with in vitro enzymatic assays. This compound is a new drug chemotype inhibiting human ACC2 with 2.8 muM IC(50) and having no effect on human ACC1 at 100 muM.

Molecular mechanism for the regulation of human ACC2 through phosphorylation by AMPK

Acetyl-CoA carboxylases (ACCs) have been highlighted as therapeutic targets for obesity and diabetes, as they play crucial roles in fatty acid metabolism. ACC activity is regulated through the short-term mechanism of inactivation by reversible phosphorylation. Here, we report the crystal structures of the biotin carboxylase (BC) domain of human ACC2 phosphorylated by AMP-activated protein kinase (AMPK). The phosphorylated Ser222 binds to the putative dimer interface of BC, disrupting polymerization and providing the molecular mechanism of inactivation by AMPK. We also determined the structure of the human BC domain in complex with soraphen A, a macrocyclic polyketide natural product. This structure shows that the compound binds to the binding site of phosphorylated Ser222, implying that its inhibition mechanism is the same as that of phosphorylation by AMPK.

Potent biphenyl- and 3-phenyl pyridine-based inhibitors of acetyl-CoA carboxylase

We report the synthesis and enzymatic evaluation of potent inhibitors of acetyl-CoA carboxylases (ACCs) containing biphenyl or 3-phenyl pyridine cores. These compounds inhibit both ACC1 and ACC2, or are moderately selective for either enzyme, depending on side chain substitution. Typical activities of the most potent compounds in this class are in the low double-digit to single-digit nanomolar range in in vitro assays using human ACC1 and ACC2 enzymes.