ACC Inhibitors Research Articles

1810-P: Triple Combination Therapy of GLP-1 Receptor Agonist Semaglutide with an FXR Agonist and AC Inhibitor Reverses Nonalcoholic Steatohepatitis (NASH) in Diet-Induced and Biopsy-Confirmed Mice

Semaglutide (SEMA), FXR agonists and ACC inhibitors are under active clinical investigation for the treatment of NASH. Here, we explored whether the effects of SEMA (0.12 mg/kg, s.c., q.d.) on NASH endpoints could be enhanced by addition of an FXR agonist (cilofexor, CILO; 30 mg/kg) and/or an acetyl-CoA carboxylase inhibitor (firsocostat analogue, ACCi; 5 mg/kg, both p.o., q.d.) in the AMLN diet-induced and biopsy-confirmed DIO-NASH mouse (n=15-16/group). After 12 weeks of treatment, SEMA and triple therapy reduced body weight 18%, being slightly enhanced in the double combination of SEMA+CILO (21%). Liver triglyceride concentration was reduced by SEMA (by 38% vs. vehicle, p<0.001). Addition of CILO or ACCi or both to SEMA further reduced liver lipid by 64%, 73% and 81%, respectively (all p<0.001 vs. SEMA). Histopathological pre-to-post NAFLD activity score (NAS) was reduced in 3/16 (19%) of vehicle controls and in all mice (100%) treated with SEMA or triple therapy. Further, the number of mice that exhibited a 3+ point improvement in NAS was improved with the triple combination (12/16; 75%) vs. SEMA alone (2/16, 13%). Likewise, the triple combination caused the greatest improvements in fibrosis stage pre-to-post (7/16, 44%, improved; 1/16 worsened), followed by the SEMA-treated group (6/16, 38% improved; 0/16 worsened) relative to vehicle (2/16, 13% improved; 7/16, 44% worsened). Collagen-1a and a-SMA immunoreactivity (% area) tended to be or were significantly improved with SEMA (4.2%; n.s., and 1.4%; p<0.001) and triple therapy (3.9%; p<0.045 and 0.9% p<0.001) vs. vehicle (6.3% and 4.6%). SEMA significantly improved NAS and fibrosis endpoints. While addition of either single agent improved liver fat reduction, triple therapy improved pre-to-post NAS and fibrosis stage relative to SEMA alone in DIO-NASH mice. These data support development of combination approaches for NASH. Disclosure J. Norlin: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. D.A. Miranda: None. J.G. Bates: Employee; Self; Gilead Sciences, Inc. N.E. Zois: Employee; Self; Gubra. S. Veidal: None. M. Feigh: None. J.L. Trevaskis: Employee; Self; Gilead Sciences, Inc. M. Latta: Employee; Self; Novo Nordisk A/S.

WZ66, a novel acetyl-CoA carboxylase inhibitor, alleviates nonalcoholic steatohepatitis (NASH) in mice.

The global prevalence of nonalcoholic steatohepatitis (NASH) increases incredibly. NASH ends up to advanced liver disease, which is highly threatening to human health. Currently, treatment of NASH is very limited. Acetyl-CoA carboxylases (ACC1/ACC2) are proved as effective drug targets for NASH. We aimed to develop novel ACC inhibitors and evaluate their therapeutic value for NASH prevention. ACC inhibitors were obtained through structure-based drug design, synthesized, screened from ACC enzymatic measurement platform and elucidated in cell culture-based assays and animal models. The lipidome and microbiome analysis were integrated to assess the effects of WZ66 on lipids profiles in liver and plasma as well as gut microbiota in the intestine. WZ66 was identified as a novel ACC1/2 inhibitor. It entered systemic circulation rapidly and could accumulate in liver. WZ66 alleviated NASH-related liver features including steatosis, Kupffer cells and hepatic stellate cells activation in diet-induced obese mice. The triglycerides (TGs) and other lipids including diglycerides (DGs), phosphatidylcholine (PC) and sphingomyelin (SM) were decreased in WZ66-treated mice as evidenced by lipidome analysis in livers. The lipids profiles in plasma were also altered with WZ66 treatment. Plasma TG were moderately increased, while the activation of SREBP1c was not detected. WZ66 also downregulated the abundance of Allobaculum, Mucispirillum and Prevotella genera as well as Mucispirillum schaedleri species in gut microbiota. WZ66 is an ideal lead compound and a potential drug candidate deserving further investigation in the therapeutics of NASH.

Inhibition of Acetyl-CoA Carboxylase by Phosphorylation or the Inhibitor ND-654 Suppresses Lipogenesis and Hepatocellular Carcinoma

The incidence of hepatocellular carcinoma (HCC) is rapidly increasing due to the prevalence of obesity and non-alcoholic fatty liver disease, but the molecular triggers that initiate disease development are not fully understood. We demonstrate that mice with targeted loss-of-function point mutations within the AMP-activated protein kinase (AMPK) phosphorylation sites on acetyl-CoA carboxylase 1 (ACC1 Ser79Ala) and ACC2 (ACC2 Ser212Ala) have increased liver de novo lipogenesis (DNL) and liver lesions. The same mutation in ACC1 also increases DNL and proliferation in human liver cancer cells. Consistent with these findings, a novel, liver-specific ACC inhibitor (ND-654) that mimics the effects of ACC phosphorylation inhibits hepatic DNL and the development of HCC, improving survival of tumor-bearing rats when used alone and in combination with the multi-kinase inhibitor sorafenib. These studies highlight the importance of DNL and dysregulation of AMPK-mediated ACC phosphorylation in accelerating HCC and the potential of ACC inhibitors for treatment.

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

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Synthesis of Spiropiperidine Lactam Acetyl-CoA Carboxylase Inhibitors

The synthesis of 4',6'-dihydrospiro[piperidine-4,5'-pyrazolo[3,4-c]pyridin]-7'(2'H)-one-based acetyl-CoA carboxylase inhibitors is reported. The hitherto unknown N-2 tert-butyl pyrazolospirolactam core was synthesized from ethyl 3-amino-1H-pyrazole-4-carboxylate in a streamlined 10-step synthesis requiring only one chromatography procedure. The described synthetic strategy provides pyrazolo-fused spirolactams from halogenated benzylic arenes and cyclic carboxylates. Key steps include a regioselective pyrazole alkylation providing the N-2 tert-butyl pyrazole and a Curtius rearrangement under both conventional and flow conditions to install the hindered amine via a stable and isolable isocyanate. Finally, a Parham-type cyclization was used to furnish the desired spirolactam. An analogous route provided efficient access to the related N-1 isopropyl lactam series. Elaboration of the lactam cores via amidation enabled synthesis of novel ACC inhibitors and the identification of potent analogues.

SCD1 Inhibition Causes Cancer Cell Death by Depleting Mono-Unsaturated Fatty Acids

Increased metabolism is a requirement for tumor cell proliferation. To understand the dependence of tumor cells on fatty acid metabolism, we evaluated various nodes of the fatty acid synthesis pathway. Using RNAi we have demonstrated that depletion of fatty-acid synthesis pathway enzymes SCD1, FASN, or ACC1 in HCT116 colon cancer cells results in cytotoxicity that is reversible by addition of exogenous fatty acids. This conditional phenotype is most pronounced when SCD1 is depleted. We used this fatty-acid rescue strategy to characterize several small-molecule inhibitors of fatty acid synthesis, including identification of TOFA as a potent SCD1 inhibitor, representing a previously undescribed activity for this compound. Reference FASN and ACC inhibitors show cytotoxicity that is less pronounced than that of TOFA, and fatty-acid rescue profiles consistent with their proposed enzyme targets. Two reference SCD1 inhibitors show low-nanomolar cytotoxicity that is offset by at least two orders of magnitude by exogenous oleate. One of these inhibitors slows growth of HCT116 xenograft tumors. Our data outline an effective strategy for interrogation of on-mechanism potency and pathway-node-specificity of fatty acid synthesis inhibitors, establish an unambiguous link between fatty acid synthesis and cancer cell survival, and point toward SCD1 as a key target in this pathway.

Discovery of small molecule isozyme non-specific inhibitors of mammalian acetyl-CoA carboxylase 1 and 2

Screening Pfizer’s compound library resulted in the identification of weak acetyl-CoA carboxylase inhibitors, from which were obtained rACC1 CT-domain co-crystal structures. Utilizing HTS hits and structure-based drug discovery, a more rigid inhibitor was designed and led to the discovery of sub-micromolar, spirochromanone non-specific ACC inhibitors. Low nanomolar, non-specific ACC-isozyme inhibitors that exhibited good rat pharmacokinetics were obtained from this chemotype.

ACC2 Is Expressed at High Levels Human White Adipose and Has an Isoform with a Novel N-Terminus

Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2) that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis. The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.

Chronic Suppression of Acetyl-CoA Carboxylase 1 in β-Cells Impairs Insulin Secretion via Inhibition of Glucose Rather Than Lipid Metabolism

Acetyl-CoA carboxylase 1 (ACC1) currently is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line, 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and by 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and the ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and tricarboxylic acid cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase protein levels were decreased by 25%, which correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor 5-(tetradecyloxy)-2-furoic acid (TOFA) to beta-cells suppressed [(14)C]glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute, suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.

N-{3-[2-(4-Alkoxyphenoxy)thiazol-5-yl]-1-methylprop-2-ynyl}carboxy Derivatives as Acetyl-CoA Carboxylase InhibitorsImprovement of Cardiovascular and Neurological Liabilities via Structural Modifications

A preliminary safety evaluation of ACC2 inhibitor 1-(S) revealed serious neurological and cardiovascular liabilities of this chemotype. A systematic structure-toxicity relationship study identified the alkyne linker as the key motif responsible for these adverse effects. Toxicogenomic studies in rats showed that 1-(R) and 1-(S) induced gene expression patterns similar to that seen with several known cardiotoxic agents such as doxorubicin. Replacement of the alkyne with alternative linker groups led to a new series of ACC inhibitors with drastically improved cardiovascular and neurological profiles.

Inhibition of Acetyl-CoA Carboxylase Isoforms by Pyridoxal Phosphate

Mammalian isoforms of acetyl-CoA carboxylase (ACC-1 and ACC-2) play important roles in synthesis, elongation, and oxidation of long-chain fatty acids, and the possible significance of ACC in the development of obesity has led to interest in the development of inhibitors. Here, we demonstrate that pyridoxal phosphate (PLP) is a linear and reversible inhibitor of ACC-1 and ACC-2. ACC from rat liver and white adipose tissue (largely ACC-1) exhibited an IC50 of approximately 200 microm, whereas ACC-2 from heart or skeletal muscle exhibited an IC50 exceeding 500 microm. ACC from rat liver was equally sensitive to PLP following extensive purification by avidin affinity chromatography. When added before citrate, PLP inhibited ACC with a Ki of approximately 100 microm, reducing maximal activity >90% and increasing the Ka for citrate approximately 5-fold but having little effect on substrate Km values. Pre-treatment with citrate increased the apparent Ki for ACC inhibition by PLP by approximately 4-fold. Inhibition of ACC was reversed by removal of PLP, either by washing or by reaction with hydroxylamine or amino-oxyacetate. ACC was irreversibly inhibited and radiolabeled, to a stoichiometry of approximately 0.4 mol[H]/mol subunit, in the presence of PLP plus [3H]borohydride. Studies with structurally related compounds demonstrated that the reactive aldehyde and negatively charged substituents of PLP contribute importantly to ACC inhibition. The studies reported here suggest a rationale to develop ACC inhibitors that are not structurally related to the substrates or products of the reaction and an approach to probe the citrate-binding site of the enzyme.

IN VITRO MORPHOGENESIS OF FIVE ORCHIDS

IN VITRO MORPHOGENESIS OF FIVE ORCHIDS

Mechanical perturbation-induced ethylene releases apical dominance in Pharbitis nil by restricting shoot growth

Mechanical perturbation (MP, rubbing) of internodes of Pharbitis nil shoots initiates release of lateral buds (LB) from apical dominance within 48 h. Evidence is presented which suggests that MP promotion of LB outgrowth is mediated by ethylene-induced restriction of main shoot growth. Ethylene production in the internodes is stimulated by MP within 2 h. Effects of MP are mimicked by treatments with 1-aminocyclopropane-1-carboxylic acid (ACC) and are negated by the inhibitors of ethylene production or action, aminoethoxy vinylglycine (AVG) and AgNO 3. The fact that effects of MP, ACC and ethylene inhibitors are observed to occur on main shoot growth at least 24 h before they are observed to occur on LB growth suggests a possible cause and effect relationship. MP also causes an increase in internode diameter. MP stimulation of ethylene production appears to be mediated by ACC synthase. The results of this study and our previous studies suggest that apical dominance may be released by any mechanism which induces ethylene restriction of main shoot growth.