Abstract
Inhibition of acetyl-CoA carboxylase (ACC), with its resultant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation, has the potential to favorably affect the multitude of cardiovascular risk factors associated with the metabolic syndrome. To achieve maximal effectiveness, an ACC inhibitor should inhibit both the lipogenic tissue isozyme (ACC1) and the oxidative tissue isozyme (ACC2). Herein, we describe the biochemical and acute physiological properties of CP-610431, an isozyme-nonselective ACC inhibitor identified through high throughput inhibition screening, and CP-640186, an analog with improved metabolic stability. CP-610431 inhibited ACC1 and ACC2 with IC50s of approximately 50 nm. Inhibition was reversible, uncompetitive with respect to ATP, and non-competitive with respect to bicarbonate, acetyl-CoA, and citrate, indicating interaction with the enzymatic carboxyl transfer reaction. CP-610431 also inhibited fatty acid synthesis, triglyceride (TG) synthesis, TG secretion, and apolipoprotein B secretion in HepG2 cells (ACC1) with EC50s of 1.6, 1.8, 3.0, and 5.7 microm, without affecting either cholesterol synthesis or apolipoprotein CIII secretion. CP-640186, also inhibited both isozymes with IC50sof approximately 55 nm but was 2-3 times more potent than CP-610431 in inhibiting HepG2 cell fatty acid and TG synthesis. CP-640186 also stimulated fatty acid oxidation in C2C12 cells (ACC2) and in rat epitrochlearis muscle strips with EC50s of 57 nm and 1.3 microm. In rats, CP-640186 lowered hepatic, soleus muscle, quadriceps muscle, and cardiac muscle malonyl-CoA with ED50s of 55, 6, 15, and 8 mg/kg. Consequently, CP-640186 inhibited fatty acid synthesis in rats, CD1 mice, and ob/ob mice with ED50s of 13, 11, and 4 mg/kg, and stimulated rat whole body fatty acid oxidation with an ED50 of approximately 30 mg/kg. Taken together, These observations indicate that isozyme-nonselective ACC inhibition has the potential to favorably affect risk factors associated with the metabolic syndrome.
Highlights
We dedicate this manuscript to the memory of John Denis McGarry, whose pioneering efforts in this area of research were an inspiration to us all, and whose guidance and support were critical to our continuing efforts and contributions to this emerging therapeutic area
In this report we describe the biochemical an physiological properties of CP-610431(Table I, inset), an isozyme-nonselective acetyl-CoA carboxylase (ACC) inhibitor identified using a 96-well multiplexed, high throughput screening paradigm and its more metabolically stable analog CP-640186, which inhibit mammalian ACC1 and acid oxidation in C2C12 cells (ACC2), reduce malonyl-CoA concentration in liver, heart, and skeletal muscle tissue, reduce fatty acid synthesis in cultured liver cells and in the liver and adipose tissue of experimental animals, increase fatty acid oxidation in cultured skeletal muscle cells and tissue slices, and reduce respiratory quotient in experimental animals
By using partially purified ACC isolated from the livers of rats that had been fasted and refed a high carbohydrate diet, and by identifying compounds that inhibited ACC activity by greater than 75%, we selected for compounds that inhibited ACC1 activity
Summary
Materials—Sodium [2-14C]acetate (56 mCi/mmol), [acetyl-3H]acetylCoA (3.3 Ci/mmol), [1-14C]palmitic acid (51 mCi/mmol), and Aquasol-2 were from PerkinElmer Life Sciences. [3H]Glycerol (20 Ci/mmol) was from American Radiochemicals After pre-incubation, 0.5-ml aliquots of assay buffer containing various concentrations of test compound were added to each flask and were followed immediately by 80 l (C2C12 cells) or 160 l (muscle strips) of a solution containing 12% ultra fatty acid-free BSA and 12.7 mM [1-14C]palmitate (3.4 Ci; 1.0 Ci/mol). In a final volume of 1.025 ml of 250 mM potassium phosphate buffer (pH 7.0), containing 2.5 mM dithiothreitol, 2.0 mM EDTA, 0.2 mM NADPH, 1 mg/ml fatty acid-free bovine serum albumin, 0.68 M [3H]acetyl-CoA (ϳ150,000 dpm/nmol), 2.0 nM malonyl-CoA standard, and either 100 l of liver extract or 200 l of skeletal muscle, heart, or adipose tissue extract, were mixed with a 10-l aliquot containing 25 milliunits of purified (as previously described (Ref. 42)) fatty acid synthetase (5 g of protein) and were incubated at 37 °C for 45 min. Oral bioavailability was calculated from the ratio of AUCs after oral and intravenous doses after normalizing for the dose
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