Acetyl-CoA carboxylase inhibition by ND-630 reduces hepatic steatosis,improves insulin sensitivity,and modulates dyslipidemia in rats

Simultaneous inhibition of the acetyl-CoA carboxylase (ACC) isozymes ACC1 and ACC2 results in concomitant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation and may favorably affect the morbidity and mortality associated with obesity, diabetes, and fatty liver disease. Using structure-based drug design, we have identified a series of potent allosteric protein–protein interaction inhibitors, exemplified by ND-630, that interact within the ACC phosphopeptide acceptor and dimerization site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes, reduce fatty acid synthesis and stimulate fatty acid oxidation in cultured cells and in animals, and exhibit favorable drug-like properties. When administered chronically to rats with diet-induced obesity, ND-630 reduces hepatic steatosis, improves insulin sensitivity, reduces weight gain without affecting food intake, and favorably affects dyslipidemia. When administered chronically to Zucker diabetic fatty rats, ND-630 reduces hepatic steatosis, improves glucose-stimulated insulin secretion, and reduces hemoglobin A1c (0.9% reduction). Together, these data suggest that ACC inhibition by representatives of this series may be useful in treating a variety of metabolic disorders, including metabolic syndrome, type 2 diabetes mellitus, and fatty liver disease.Fatty acid metabolism dysregulated through elevated fatty acid synthesis (FASyn), impaired fatty acid oxidation (FAOxn), or both is a hallmark of various metabolic disorders, including insulin resistance, hepatic steatosis, dyslipidemia, obesity, metabolic syndrome (MetSyn), and nonalcoholic fatty liver disease (NAFLD), that can lead to the development of type 2 diabetes (T2DM), nonalcoholic steatohepatitis (NASH), and atherosclerotic vascular disease (1–6). Altered fatty acid metabolism also is a hallmark of cancer and contributes to the abnormal and sustained cellular proliferation of malignancy (7, 8). Therefore inhibition of FASyn and/or stimulation of FAOxn have the potential to affect these maladies favorably.As a result of its unique position in intermediary metabolism, pharmacologic inhibition of acetyl-CoA carboxylase (ACC) offers an attractive modality for limiting FASyn in lipogenic tissues while simultaneously stimulating FAOxn in oxidative tissues (1, 9). ACC catalyzes the ATP-dependent carboxylation of acetyl-CoA to form malonyl-CoA, the rate-limiting and first committed reaction in FASyn (1, 9–11). This conversion proceeds in two half-reactions, a biotin carboxylase (BC) reaction and a carboxyltransferase (CT) reaction (1, 9–11). ACC activity is tightly regulated through a variety of dietary, hormonal, and other physiological responses including feed-forward activation by citrate, feedback inhibition by long-chain fatty acids, reversible phosphorylation and inactivation by AMP-activated protein kinase (AMPK), and modulation of enzyme production through altered gene expression (1, 9–12).ACC exists as two tissue-specific isozymes that are encoded by separate genes and display distinct cellular distributions (10, 13, 14). ACC1 is a cytosolic enzyme present in lipogenic tissues (liver, adipose); ACC2 is a mitochondrially associated isozyme present in oxidative tissues (liver, heart, skeletal muscle) (10, 15). In the liver, malonyl-CoA formed in the cytoplasm by ACC1 is used primarily for FASyn and elongation (1), whereas malonyl-CoA formed at the mitochondrial surface by ACC2 acts primarily to regulate mitochondrial FAOxn (1) through allosteric inhibition of carnitine palmitoyltransferase-1 (16). This functional compartmentalization results from a combination of synthesis proximity and the rapid action of malonyl-CoA decarboxylase (1). In the heart and skeletal muscle, which lack ACC1 and thus have a limited capacity for FASyn, the malonyl-CoA formed by ACC2 functions primarily to regulate FAOxn (1). Adipose tissue primarily contains ACC1 to support FASyn in that tissue (1).Over the past two decades numerous lines of evidence have emerged that strongly support the concept that direct inhibition of ACC is an important therapeutic target. Initial studies with the long-chain fatty acid analog 5-(tetradecyloxy)-2-furancarboxylic acid (17, 18) and the isozyme-nonselective, active site-directed ACC inhibitor CP-640186 (1, 19) have demonstrated the potential for direct ACC inhibition to affect favorably a plethora of metabolic disorders. These pharmacologic studies have been supported further through genetic manipulation of ACC, including studies with ACC2-knockout mice (20, 21), ACC antisense oligonucleotides (22), TRB3 transgenic mice that have increased rates of ACC degradation (23), and mice with alanine-knockin mutations in the AMPK phosphorylation sites on ACC1 and ACC2 that render them constitutively active (24). Furthermore, studies with combined ACC1 and ACC2 antisense oligonucleotides (22) and with isozyme-specific and/or tissue-specific ACC-knockout mice (25, 26) have provided strong evidence that dual inhibition of ACC1 and ACC2 is superior to the inhibition of either isozyme alone.A large number of isozyme-nonselective ACC inhibitors have been identified that interfere directly with ACC catalysis through interaction within the CT domain of the enzyme and have been shown to be potent in vitro and efficacious in vivo (1, 9, 19, 27–30). Indeed, in early clinical trials one such inhibitor recently has been shown to reduce FASyn and to stimulate FAOxn after a single oral dose (29). However, the regions of the enzyme to which these inhibitors interact are very hydrophobic (9, 11, 31–33), and thus these inhibitors lack optimal pharmaceutical properties. In contrast, the dimerization site of the enzyme, located on the BC domain, is a shallow, hydrophilic pocket that is the site to which both the phosphopeptide of ACC that is phosphorylated by AMPK and the natural products fungicide soraphens bind to prevent dimerization and inhibit enzymatic activity (9, 34–36). We therefore hypothesized that an allosteric inhibitor that binds to this region of the enzyme would exhibit superior physicochemical properties, would be highly selective relative to other carboxylases because this site is not conserved among the mammalian carboxylases (37), and would mimic the physiological inhibition of ACC by AMPK.In this report, we describe use of structure-based drug design (SBDD) to identify a unique series of potent and efficacious allosteric protein–protein interaction inhibitors that interact within the ACC subunit dimerization and phosphopeptide acceptor site to prevent dimerization and inhibit the enzymatic activity of both ACC isozymes. We also demonstrate that ND-630, a representative analog of this series that exhibits favorable pharmaceutical properties, is highly effective in reducing hepatic steatosis, improving insulin sensitivity, and favorably affecting dyslipidemia in rats with diet-induced obesity (DIO) and in Zucker diabetic fatty (ZDF) rats, suggesting its utility in treating a variety of metabolic disorders.