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Recent Advances in the Development of Acetyl-CoA Carboxylase (ACC) Inhibitors for the Treatment of Metabolic Disease Miniperspective Matthew P. Bourbeau* and Michael D. Bartberger Department of Medicinal Chemistry, and Department of Molecular Structure and Characterization, Amgen, Inc., 1 Amgen Center Drive, Thousand Oaks, California 91320, United States ABSTRACT: The development of acetyl-CoA carboxylase (ACC) inhibitors for the treatment of metabolic disease has been pursued by the pharmaceutical industry for some time. A number of recent disclosures describing potent ACC inhibitors have been reported by multiple research groups. Unlike many prior publications in this area, more recent publications contain a significant amount of in vivo efficacy data generated by long-term experiments in rodent models of metabolic disease. Additionally, one compound has been advanced to human clinical studies. The results from these studies should allow researchers to better gauge the potential utility of ACC inhibition for the treatment of human disease.
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INTRODUCTION The acetyl-CoA carboxylases (ACCs) are enzymes that are involved in the synthesis and oxidation of fatty acids and have been targeted as potential intervention points for the treatment of metabolic diseases such as type 2 diabetes and dyslipidemia. The ACC protein structure consists of three function domains: the biotin carboxylase domain (BC), the biotin carboxyl carrier protein domain (BCCP), and the carboxyl transferase domain (CT) (Figure 1).1 There are two characterized isoforms of mammalian ACC, both of which catalyze the conversion of acetyl-CoA to malonylCoA.2,3 The first isoform, known as ACC1, is located in the cytosol and is primarily expressed in lipid rich tissue (liver, adipose). It is responsible for the rate limiting initiation of de novo fatty acid synthesis. In contrast, the second ACC isoform, ACC2, is imbedded in the mitochondrial membrane and is primarily found in oxidative tissue (muscle).4,5 ACC2 serves as a negative regulator of fatty acid uptake by the mitochondria via the inhibition of carnitine palmitoyltransferase I (CPT1), the protein that is responsible for the conjugation of free fatty acyls to carnitine for transfer across the mitochondrial membrane for subsequent β-oxidation. Thus, inhibition of the ACCs might be expected to reduce both fatty acid synthesis while simultaneously increasing fatty acid oxidation.
in complex with coenzyme A (CoA, 1) demonstrated that the active site resides at the dimeric interface of the N and C domains (wheat and green, respectively; Figure 3a).6 Co-crystal structures of the herbicides 2 (haloxyfop) and 3 (diclofop) (orange and yellow, respectively, in Figure 2a) were also found to bind at the dimeric interface, from which the basis of ACC inhibition by these herbicides (partial occlusion of the CoA binding cleft) could be determined.7 Compound 4 (CP-640186), an ACC1/ ACC2 dual inhibitor developed by Pfizer that is noncompetitive with acetyl-CoA, has also been crystallized in the yeast ACC CT domain.8 The anthracenyl moiety of 4 is sandwiched between α6 and α6′ helices of the N and C domains of ACC2 near the dimer interface, yet (in contrast to herbicide binding) does not overlap with the region corresponding to CoA binding, consistent with the competitive kinetic results. It was proposed that 4 may occupy the region utilized by biotin, the carboxylated form serving as the carboxyl source in the transformation of acetyl- to malonyl-CoA. Further cocrystal structures published by the Tong group of herbicides 5 (tepraloxydim) and 6 (pinoxaden) demonstrate a greater degree of occlusion of the CoA binding cleft (Figure 3b) compared to haloxyfop and diclofop, yet with less conformational change than that required for the latter herbicides.9,10 Finally, the structural basis of ACC inhibition by the potent polyketide natural product soraphen A1α (7) has been determined.11 Genetic, biochemical, and ultimately structural studies have demonstrated that, in stark contrast to that of the binding of the previous described ACC inhibitors, 7 binds to the BC domain of ACC (Figure 4), likely disrupting BC
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STRUCTURAL CONSIDERATIONS A number of crystallographic studies from the research group of Tong6−11 have uncovered the structural basis for inhibition by a diverse array of ligands and have shown that potent ACC inhibition can occur via binding to either the CT or BC domains (Figure 2). The crystal structure of the CT domain of yeast ACC © XXXX American Chemical Society
Received: May 5, 2014
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Figure 1. Schematic depicting various ACC domains and their functions.
Figure 2. Small molecules cocrystallized with ACC.
Figure 3. (a) Alignment of cocrystal structures of 1 (white, PDB 1OD2), 2 (orange, PDB 1UYS), 3 (yellow, PDB 1UYR), and 4 (cyan, PDB 1W2X). For clarity, only the backbone structure of coenzyme A-bound ACC CT dimer (N-domain in wheat, C-domain in green) is depicted, although in some cases significant side chain conformational change occurs (not shown) upon ligand binding. Backbone transparency was used to indicate the foreground. (b) Alignment of cocrystal structures of 5 (magenta, PDB 3K8X) and 6 (yellow, 3PGQ) with 1, 2, 3, and 4. For clarity, only the backbone structure of coenzyme A-bound ACC CT dimer (N-domain in wheat, C-domain in green) is depicted, although in some cases significant side chain conformational change occurs (not shown) upon ligand binding. Backbone transparency was used to indicate the foreground. Compounds 1, 2, and 3 were rendered transparently to highlight 5 and 6 binding, spanning the respective binding pockets.
dimerization. The crystallographically demonstrated existence of multiple ligand binding sites, involving more than one domain of
the ACCs, underscores the availability of multiple potential avenues for activity inhibition by small molecules. B
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Wakil’s case, the biotin binding domain of ACC2 was replaced with hypoxanthine phosphorylribosyltransferase (HPRT) expression cassette, whereas Cooney and Lowell ACC−/− mice utilized LoxP methodology to delete the exon containing the biotin binding domain of ACC2 entirely. Lowell also postulates that Wakil’s ACC2−/− mice may contain mutant, catalytically inactive ACC2 protein that may exert a dominant negative effect on ACC1, resulting in a phenotype that is not entirely ACC2 dependent. Additionally, it is possible that mouse background strain (different for all three ACC2−/− models) may have had a confounding influence over the whole animal metabolic phenotype. Efforts to generate ACC1 knockout mice (ACC1−/−) met with initial failure, as the knockout proved to be embryonically lethal.18 However, two liver specific ACC1 knockout mouse models (LACC1−/−) have been reported by the groups of Wakil and Kusunoki.19,20 Liver samples from the LACC1−/− mice in Wakil’s study showed significantly reduced malonyl-CoA levels and ACC activity relative to WT mice, along with a significant reduction in fatty acid synthesis. On the other hand, liver samples from the LACC1−/− mice generated by Kusunoki’s group showed no differences in malonyl-CoA levels or fatty acid synthesis relative to WT mice. Liver samples from Kusunoki’s LACC1−/− mice did, however, show increased levels of both ACC2 mRNA and protein expression, suggesting that ACC2 was being up-regulated to accommodate the deletion of ACC1. As was the case with the various ACC2−/− mice, there is no obvious explanation for the different phenotypes seen in Wakil and Kusunoki’s LACC1−/− mice, although the same sort of rationalizations used to offer possible explanations for the differences seen in the different ACC2−/− mouse strains could apply here as well. No attempts to produce ACC2−/−/ACC1−/− mice have been reported. In addition to mouse knockout studies, there is some human data to suggest that ACC inhibition may provide a therapeutic benefit. Healthy volunteers placed on a 3 month exercise program showed decreases in ACC2 mRNA levels and increases in fatty acid oxidation during the course of the study.21 Obese and type 2 diabetic patients have been shown to have increased malonyl-CoA levels and ACC2 activity.22 Thiazolidinedione
Figure 4. Cocrystal structure of 7 (white, PDB 1W96) bound to the BC domain of yeast ACC (green).
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IN VIVO PHARMACOLOGY ACC activity and function have been studied in a number of different mouse knockout models. The first such mouse model reported was an ACC2 knockout (ACC2−/−), described in a series of publications from Wakil and co-workers. 12−15 Compared to wild type (WT) mice, these ACC2−/− mice showed a significant reduction in malonyl-CoA levels in skeletal muscle and heart tissue, along with corresponding increases in fatty acid oxidation in these same tissues. Wakil’s ACC2−/− mice had increased whole body energy expenditure relative to WT mice and showed improved insulin and lipid profiles when placed on a high fat/high calorie diet. Subsequent to Wakil’s work, the groups of Cooney and Lowell reported ACC2−/− mice generated by an alternative methodology to that used by Wakil’s group.16,17 Conney’s ACC2−/− mice exhibited reduced malonyl-CoA levels in heart (60%) and muscle (50%) tissues relative to WT mice, resulting in increased muscle and whole body fatty acid oxidation. Lowell’s ACC2−/− mice showed an even more modest reduction of malonyl-CoA levels in heart (30%) relative to WT mice, with no other significant metabolic changes observed. It is difficult to reconcile the significant differences in the observed phenotypes of these three ACC2−/− mouse strains. One possible explanation is that the cloning methods used by the different laboratories to generate the ACC2−/− mice were not identical. In
Figure 5. ACC inhibitors reported by Taisho. C
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treatment has been shown to reverse this effect. Additionally, it is thought that the efficacy of antidiabetic agent metformin may be due in part to its activation of adenosine monophosphateactivated protein kinase (AMPK), which serves to inhibit ACC2 activity in vivo.23 Driven largely by the initially promising ACC2−/− mouse studies from Wakil’s lab,12−15 there has been a significant amount of effort directed toward the development of ACC inhibitors for the treatment of metabolic diseases.24−27 At the time this topic was last reviewed,25 there had been a modest amount of longterm efficacy data reported related to the effect of ACC inhibition in improving metabolic parameters.28 This review aims to cover subsequent relevant literature, including reports of several longterm efficacy studies with newly developed ACC inhibitors. Taisho has previously reported a series of ACC1/ACC2 dual inhibitors (Figure 5).29 The most thoroughly profiled of these was compound 8, which inhibited ACC enzymatic activity [human ACC1 (hACC1) IC50 = 101 nM, human ACC2 (hACC2) IC50 = 23 nM]. Compound 8 also increased fatty acid oxidation (EC50 = 580 nM) and decreased fatty acid synthesis (IC50 = 340 nM) in HepG2 cells. A subsequent paper detailed efforts examining changes to the 2,6-diarylpyridine portion of the 8, focusing on increasing the size of one of the aryl groups.30 This resulted in the synthesis of compounds such as indazole 9, which inhibited hACC1/2 enzymatic activity with an IC50 of 33 nM. (Note: For the bulk of these studies, Taisho utilized partially purified human hACC enzyme that contained a mixture of hACC1 and hACC2.) Further efforts to replace the 2,6-diarylpyridine functional group led to the discovery of a benzothiophene urea class of ACC inhibitors.31 In particular, compound 10 inhibited hACC1/2 enzymatic activity with IC50 = 74 nM. It was then found that the bis-piperidine amide portion of 10 could be truncated, resulting in tert-butyl carbamate 11, a compound that showed improved hACC1/2 enzymatic inhibition (IC50 = 24 nM) and inhibited fatty acid synthesis in HepG2 cells (IC50 = 79 nM). Further work to improve the stability of the tert-butyl carbamate group to acidic conditions resulted in the synthesis of compound 12, which was profiled extensively. Compound 12 inhibited human and rat ACC1/2 (rACC1/2, partially purified enzyme containing a mixture of rACC1 and rACC2) enzymatic activity (hACC1 IC50 = 192 nM, hACC1 IC50 = 95 nM, rACC1/2 IC50 = 32 nM). Compound 12 also increased fatty acid oxidation (EC50 = 370 nM) and decreased fatty acid synthesis (IC50 = 60 nM) in HepG2 cells. Treatment of Sprague−Dawley (SD) rats with compound 12 (10 mg/kg, po) resulted in a 74.6% decrease in liver fatty acid synthesis 1 h postdose. Encouraged by these results, compound 12 was then dosed for 12 days po (oral), b.i.d. (twice daily), in SD rats placed on a high fructose diet. Animals dosed with 12 had significantly reduced plasma and liver triglyceride levels relative to the control group, with a minimum effective dose of 3−10 mg/ kg compound 12. In addition to the observed triglyceride effects, animals dosed with compound 12 showed significant skin irritation around the nose and toes. As lipids are an essential component of normal skin, the authors stated that they could not rule out the possibility that the skin irritation was a mechanism based effect of ACC inhibition of de novo fatty acid synthesis. Sanofi-Aventis has reported the development of a series of ACC1/ACC2 dual inhibitors (Figure 6).32 Starting from 1,3dioxane 13, a high throughput screening hit that was shown to inhibit hACC2 (IC50 = 630 nM) but was inactive on hACC1, rat ACC2 (rACC2), and rat ACC1 (rACC1), medicinal chemistry efforts led to the synthesis of compound 14, which showed
Figure 6. ACC inhibitors reported by Sanofi-Aventis.
enzymatic inhibition of all ACC isoforms tested (hACC2 IC50 = 30 nM, hACC1 IC50 = 190 nM, rACC2 IC50 = 400 nM, rACC1 IC50 = 170 nM). Compound 14 caused a dose dependent increase in fatty acid oxidation in human hepatocytes and possessed suitable in vivo pharmacokinetic (PK) properties for further profiling (plasma PK studies in Wistar rats: iv (3 mg/kg) CL = 1.6 L h−1 kg−1, Vss = 2.6 L/kg; po (10 mg/kg) CMax = 3.5 μmol/L, TMax = 2 h, AUC = 19 μmol·h/L, t1/2 = 1.8). Female Wistar rats dosed with 50 mg/kg compound 14 followed by the administration of triglycerides experienced a greater reduction in respiratory quotient (RQ) during the light phase, indicating that compound 14 was simulating fatty acid oxidation in vivo. Additionally, when obese female Zucker diabetic fatty (ZDF) rats were dosed orally with 30 mg/kg compound 7 for 3 days q.d. (once daily), a decrease in triglyceride levels was observed in the treated animals relative to the control group. A subsequent publication examined the effect of treatment with compound 14 over a longer dosing period.33 Two sets of experiments were run using diet induced obese (DIO) female C57BL6 mice treated with compound 14. In the first study, the DIO mice were prepared by treatment with a high-fat diet for 24 weeks. Compound 14 was then dosed for 29 days q.d. while maintaining the high-fat diet. Animals treated with 14 showed no change in body weight or food consumption relative to the control group on a high-fat diet. Hepatic malonyl-CoA levels were decreased, and total ketone bodies, a marker of increased fatty acid oxidation, were increased in the mice treated with 14, indicating that there was an increase in fatty acid oxidation corresponding to malonyl-CoA reduction. Nonesterified fatty acid levels were also decreased in mice treated with 14 relative to the control group, but there were no changes observed in plasma triglycerides, hepatic triglycerides, or glucose tolerance observed between mice treated with compound 14 and the control group. The second study was designed to determine if treatment with compound 14 could prevent the development of metabolic changes associated with a high-fat diet. To this end, C57BL6 mice were fed a high-fat diet over a 6-week period with or without administration of compound 14. A control group of mice fed standard chow was also included in this study. Mice placed on the high-fat diet showed significant increases in body weight and body fat, as well as increased hepatic triglycerides, blood glucose, and cholesterol levels relative to mice on standard chow. The high-fat mice also showed impaired glucose tolerance. Mice on the high-fat diet that were treated with compound 14 showed increase plasma ketone bodies and increased liver weight relative to high-fat mice that were not administered 14; however, there were no differences observed in body weight gain, hepatic triglyceride accumulation, or glucose tolerance. In addition to the studies in DIO mice, an 8-day study in female ZDF rats dosed with compound 14 was reported. As seen in the previous study,31 a decrease in hepatic triglyceride levels was observed at day 3 in animals treated with compound 14 relative to the control group. However, at day 8 of the study, no such differentiation was observed, suggesting that the triglyceride lowering was a transient effect. There was an increased serum level of ketone bodies and decreased serum level of nonesterified D
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Figure 7. ACC inhibitors reported by AstraZeneca.
significantly decreased protein binding (fraction unbound of 3.4 and 3.3, respectively) relative to 18, although rat iv clearance was still fairly high for both compounds (CL of 32 and 70 mL min−1 kg−1, respectively). Compounds 20 and 21 showed cross-species reactivity in rat ACC enzyme assays (20, rACC2 IC50 = 190, rACC1 IC50 = 460 nM; 21, rACC2 IC50 = 150, rACC1 IC50 = 250 nM) and were dosed in obese ZDF rats via iv infusion. There was an approximately 50% reduction in hepatic malonyl-CoA levels, observed in the rats treated with compounds 20 and 21 relative to the control group, corresponding to unbound plasma concentrations 2- to 3-fold above the in vitro IC50 for rat ACC2 inhibition. No other in vivo parameters were measured in this study. Takeda’s work to develop ACC inhibitors was initiated by the examination of a known inhibitor 4 published by Pfizer (4, hACC2 IC50 = 34 nM, hACC1 IC50 = 550 nM) (Figure 8).36,37 Compound 4 has been shown crystalographically to bind to the CT domain of ACC (Figure 3).8 Modeling of 4 with hACC2 suggests that the two amide carbonyl oxygens make key interactions with ACC, the morpholine amide with Gly2162 and the anthracene amide with Glu2230.38 The Takeda group hoped
fatty acids in the compound 14 treated group on both day 3 and day 8, suggesting that fatty acid oxidation was elevated for the entire dosing period. The paper concludes that despite the ability of compound 14 to lower malonyl-CoA levels in rodent models of metabolic disease, there was no evidence of any improvement observed in hepatic lipid levels, glucose tolerance, or body weight relative to control animals in these studies. AstraZeneca has reported the development of a series of ACC1/ACC2 dual inhibitors that was initiated by the identification of screening hits 15 and 16 (15, hACC2 IC50 = 2.5 μM; 16, hACC2 IC50 = 3.6 μM) (Figure 7).34 Examination of the structures of 15 and 16 suggested that these compounds might share an overlapping pharmacophore and that hybrid analogs combining features from both compounds might show increased ACC inhibitory activity. This resulted in the synthesis of compounds 17 and 18, which did in fact show improved enzyme inhibition (17, hACC2 IC50 = 609 nM; 18, hACC2 IC50 = 210 nM). Compound 18 was further profiled to see if it might be of use as an in vivo tool. Unfortunately, compound 18 demonstrated very high human plasma protein binding (fraction unbound of
