A novel direct homogeneous assay for ATP citrate lyase

ATP citrate lyase (ACL) catalyzes the production of cytosolic acetyl-CoA and oxaloacetate from citrate. In mammals, the formation of acetyl-CoA is an essential step for the de novo synthesis of fatty acid (FA) and cholesterol for converting the carbohydrate carbon energy source into lipids. Hence, it has been thought that ACL inhibition would be beneficial for the treatment of obesity and dyslipidemia through the simultaneous inhibition of endogenous synthesis of FA and cholesterol. Several citrate analogs have been reported as ACL inhibitors in the literature. Hydroxycitrate (HCA), a potent competitive inhibitor of ACL (Ki = 150 nM) (1), has been shown to inhibit the synthesis of both FA and cholesterol and increase the LDL receptor activity (2). In vivo administration of HCA reduced plasma cholesterol and triglycerides in rats (3). Of note, HCA is also thought to be the active ingredient of CitriMax, a nutritional supplement used in controlling human body weight (4). In obese rodent models, HCA reduced food intake and body weight gain (5). Weight loss was attributed to selective lowering of body fat with no change in body protein composition. In addition, SB-201076, another citrate analog with a reported Ki of 1.0 μM, has been tested in both rats and dogs. SB-204990, the cell-permeable γ-lactone prodrug of SB-201076, inhibited cholesterol and fatty acid synthesis in a dose-dependent manner in HepG2 cells (91% and 82%, respectively) and rats (76% and 39%, respectively). When administered for 1 week, SB-204990 decreased plasma cholesterol by 46% and plasma triglycerides by 80% in rats. SB-204990 (dosed at 25 mg/kg for 2 weeks) also decreased plasma cholesterol up to 23% and plasma triglycerides up to 38% in dogs (6). Recently, Li et al. (7) described a 2-hydroxy-N-arylbenzenesulfonamides class of compounds as ACL inhibitors that structurally deviate from citrate and demonstrate higher cell permeability and in vivo bioavailability. Chronic administration of Compound 9 (or BMS-303141), the leading inhibitor in this class, in high-fat–fed mice reduced weight gain and lowered plasma cholesterol, triglycerides, and glucose. All these data are consistent with the hypothesis that ACL might be an attractive target for the treatment of metabolic disorders, including obesity and dyslipidemia. Traditionally, ACL activity is measured using coupling enzymes such as malic dehydrogenase (MDH) (8) or chloramphenicol acetyl transferase (9). This coupling approach, however, is an indirect measure of ACL enzyme activity, and detailed enzymatic features or enzyme/inhibitor interactions may be missed. To avoid the shortcomings inherent in the coupled enzymatic assays, we describe in this report a novel procedure to directly measure ACL enzyme activity in 384-well plate format suitable for high-throughput screening.