| Assay Method Information | |
| | In Vitro Acetyl-CoA Carboxylase (ACC) Inhibition Assay |
| Description: | An exemplary procedure for the in vitro ACC inhibition assay, which can be used to determine the inhibitory action of compounds of the invention toward either ACC1 or ACC2, follows. The ADP-Glo Kinase Assay kit from Promega is used. The ADP-Glo Kinase Assay is a luminescent ADP detection assay to measure enzymatic activity by quantifying the amount of ADP produced during an enzyme reaction. The assay is performed in two steps; first, after the enzyme reaction, an equal volume of ADP-Glo Reagent is added to terminate the reaction and deplete the remaining ATP. Second, the Kinase Detection Reagent is added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. Luminescence can be correlated to ADP concentrations by using an ATP-to-ADP conversion curve. The detailed procedure is as follows. 50 μL of the compound being tested (600 uM in DMSO) is added to a 384-well dilution plate. The compound is diluted 1:3 in succession in DMSO for each row for 11 wells. 0.5 μL ACC2 working solution is added to 384-well white Optiplate assay plate. 0.5 μL diluted compound solution in each column from step 2 is added to the assay plate, each row containing 2 replicates. For the last 2 rows, add 0.5 μL negative control (DMSO) in one row and 0.5 μL positive control (compound I-97) in the other. The plates are incubated at room temperature for 15 minutes. 5 μL substrate working solution is added to each well to initiate reaction. Final ACC2 reaction concentrations consist of: 5 nM ACC2, 20 μM ATP, 20 μM acetyl-CoA, 12 mM NaHCO3, 0.01% Brij35, 2 mM DTT, 5% DMSO, test compound concentrations: 30 μM, 10 μM, 3.33 μM, 1.11 μM, 0.37 μM, 0.123 μM, 0.0411 μM, 0.0137 μM, 0.00457 μM, 0.00152 μM, and 0.00051 μM. Plates are incubated at room temperature for 60 minutes. 10 μL ADP glo reagent is added. Plates are incubated at room temperature for 40 minutes. 20 μL kinase detection reagent is added. Plates are incubated at room temperature for 40 minutes, then read on a Perkin Elmer EnVision 2104 plate reader for luminescence as Relative Light Units (RLU). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |