A novel direct homogeneous assay for ATP citrate lyase.

ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes the synthesis of acetyl-CoA and oxaloacetate using citrate, CoA, and ATP as substrates and Mg(2+) as a necessary cofactor. The ACL-dependent synthesis of acetyl-CoA is thought to be an essential step for the de novo synthesis of fatty acids and cholesterol. For this reason, inhibition of ACL has been pursued as a strategy to treat dyslipidemia and obesity. Traditionally, ACL enzyme activity is measured indirectly by coupling to enzymes such as malate dehydrogenase or chloramphenicol acetyl transferase. In this report, however, we describe a novel procedure to directly measure ACL enzyme activity. We first identified a convenient method to specifically detect [(14)C]acetyl-CoA without detecting [(14)C]citrate by MicroScint-O. Using this detection system, we devised a simple, direct, and homogeneous ACL assay in 384-well plate format that is suitable for high-throughput screening. The current assay consists of 1) incubation of ACL enzyme with [(14)C]citrate and other substrates/cofactors CoA, ATP, and Mg(2+), 2) EDTA quench, 3) addition of MicroScint-O, the agent that specifically detects product [(14)C]acetyl-CoA, and 4) detection of signal by TopCount. This unique ACL assay may provide more efficient identification of new ACL inhibitors and allow detailed mechanistic characterization of ACL/inhibitor interactions.

. We speculate that the reason MicroScint-O specifi cally detects [ 14 C]acetyl-CoA, but not [ 14 C]citrate, is because of its special design in cocktail physical chemical characteristics that allow it to solubilize acetyl-CoA better than the more hydrophilic citrate. According to the manufacturer Perki-nElmer, the patented MicroScint-O cocktail is only suitable for counting nonpolar hydrophobic samples. The cocktail does not contain surfactants; therefore, Micro-Scint-O is not miscible with water and is unable to detect aqueous samples. in this report a novel procedure to directly measure ACL enzyme activity in 384-well plate format suitable for highthroughput screening.

Materials
We obtained [ 14 C]citric acid from GE Healthcare and Micro-Scint-O and 384-well PolyPlate from Perkin Elmer. All other reagents are the highest purity from Sigma.

Human ACL expression and purifi cation
Recombinant human ACL was expressed and purifi ed according to Lord et al. ( 10 ) with minimal modifi cations. Full-length human ACL coding region was cloned and expressed in Baculovirus Bac-N-Blue expression system (Invitrogen). Sf9 cells were infected by the recombinant baculovirus at multiplicity of infection of 5 for 55 h. The infected cells were resuspended and sonicated in Buffer A (10 mM HEPES, pH 7.2, 280 mM sucrose, 2 mM DTT, 0.24 mM EGTA, and 1 mM EDTA) supplemented with 1 mM PMSF, 1 mM leupeptin, and 1 mM pepstatin A to break the cells. The lysate was centrifuged at 45,000 rpm in a Ti60 rotor for 1 h. The supernatant was precipitated with 40% ammonium sulfate and the resultant pellet was resuspended and dialyzed against Buffer B (20 mM Tris, pH 8.0, 1 mM MgCl 2 , 10 mM ␤ -mercaptoethanol, 0.1 mM EGTA). The dialysate was then purifi ed by ion exchange DEAE chromatography followed by gel-fi ltration Sephacryl S300 chromatography in Buffer C (50 mM Tris, pH 8.0, 50 mM NaCl, 2 mM DTT, 1 mM MgCl 2 , 10% glycerol). The active peaks were pooled and stored in Buffer C at Ϫ 80°C for future use. The recombinant ACL purifi ed with this method is about 90% pure as assessed by SDS-PAGE ( supplementary Fig. I ).

Steady-state kinetic parameters of ACL
To determine the steady-state kinetic parameters of ACL by the direct assay, substrate concentration-dependent enzyme reactions were conducted as shown in Fig. 4 . Table 1 summarizes the K m values: 73.8 ± 11.4 M for citrate, 4 ± 2 M for CoA, and 47 ± 17 M for ATP. Although the K m value for citrate derived from the current direct assay was similar compared with that determined by the ACL/MDH coupled assay, K m values of CoA and ATP were about 3-fold less than those determined by the coupled assay.

Assay statistics
To evaluate the consistency and reproducibility of the direct ACL assay, we adopted a statistical approach. The assays were performed in two separate plates on each day for two consecutive days. Each plate had 48 wells containing substrates, reagents, and human ACL enzyme with the The signal was shown to be stable (80% of the signal remained even after 5 days). For consistency, all remaining studies described were analyzed after an overnight incubation with shaking at room temperature.

Effect of carrier proteins
As described above, our initial data indicated that the MicroScint-O method directly and selectively measured [ 14 C]acetyl CoA in a reproducible manner. However, during the enzyme titration experiment, we found that ACL started to lose linearity when the ACL concentration dropped below 100 ng/reaction (data not shown). One possibility is that the ACL enzyme may not be stable at low concentrations. Alternatively, nonspecifi c protein adsorption to plastic surfaces may cause loss of activity. This problem was not manifested in coupled assay systems, because high concentrations of MDH or chloramphenicol acetyl transferase coupling enzyme may provide a stabilizing protein environment that prevents the loss of ACL activity. This hypothesis is supported by data in Fig. 2 . In the absence of carrier protein activity produced by 50 ng ACL is less than one-half of that produced by 100 ng ACL (data beyond 2 h of incubation). In addition, after 3 h, no increase of activity by 50 and 100 ng ACL was observed, indicating that the enzyme was inactivated. Notably, nonspecifi c carrier proteins ␤ -lactoglobulin and BSA, at either 0.01% or 1%, signifi cantly increased the activity produced by 50 ng ACL. Further, addition of these agents also helped sustain enzyme activity beyond 3 h at 37°C. Between these two carriers, ␤ -lactoglobulin appeared to possess a better protective result, as 0.01% of ␤ -lactoglobulin produced the same ACL stabilizing effects as 1% lactoglobulin and BSA. That there is no difference in activity between 0.01% and 1% lactoglobulin suggests saturation of the stabilizing effect. Glycerol at 1% did not produce any appreciable protective effect. Based on these results, 0.01% ␤ -lactoglobulin was selected as the routine carrier protein for the direct ACL assay in all remaining studies.

Concentration-and time-dependent ACL activity
To further determine the optimal ACL reaction conditions, ACL concentrations and reaction time were systematically titrated. As shown in Fig. 3 , the signal increased linearly with increasing amounts of ACL and reaction time. When using 30 ng ACL, the reaction was linear up to 3 h.  than 9, indicating that the direct ACL assay is both very robust and highly reproducible.

Effect of reaction temperature, DMSO, and assessment of ACL inhibitor
We also compared temperature effects on the current direct assay. At 20°C, ACL produced one-half of the signal compared with 37°C. However, the signal-to-noise values were both approximately 15.
For inhibitor screening purpose, compounds were routinely dissolved in 100% DMSO with further dilution to 1% or lower in the fi nal assay. DMSO up to 5% had no signifi cant effect on ACL activity.
To assess if the current assay consistently reports inhibitory potencies of a given ACL inhibitor, we tested BMS-303141 [or compound 9 in ( 7 )] against ACL. As shown in Fig. 5 , when tested on separate days and in different plates, the shape of the concentration-inhibition curves and the degree of inhibition by BMS-303141 were highly reproducible. The IC 50 values were determined to be 0.80, 1.00, 1.23, and 0.74 M using four independent curves. The quenching EDTA added before the reaction to defi ne the background level or the minimal signal level, and 48 wells containing all of the above except that the quenching EDTA was added after the enzymatic reaction to defi ne the maximal signal level. The statistics for each plate were calculated independently and tabulated in Table 2 according to methods described in online supplementary data Table I . The current direct ACL assay has a Z' greater than 0.6, signal/noise and signal/background values greater

CONCLUSIONS
The work described here presents a novel assay for the direct measurement of ACL enzymatic activity. The assay design utilizes the unique ability of MicroScint-O to selectively detect [ 14 C]acetyl CoA signal from [ 14 C]citrate. Table 3 summarizes the major differences between the current direct assay and the traditional ACL-MDH coupled assays. The direct assay offers several advantages: 1 ) it is conducted in 384-well plate format with lower assay volume and 10-fold less ACL enzyme; and 2 ) the assay is homogeneous where all the reagents are added and signals are detected in the same reaction vessel. In addition, BMS-303141, a BMS ACL inhibitor, exhibited a consistent and classic sigmoidal inhibition pattern using this assay. The statistics parameters of the assay performance further demonstrate that it is a robust and reliable assay amenable to high-throughput screening.