| Assay Method Information | |
| | In Vitro Assay of Acetyl-CoA Carboxylase (ACC) Inhibition |
| Description: | An exemplary procedure for the in vitro ACC inhibition assay, which can be used to determine the inhibitory action of the compounds of the invention toward either ACC1 or ACC2, is as follows. The ADP-Glo™ kinase assay kit from Promega was 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 was performed in two steps; first, after the enzyme reaction, an equal volume of ADP-Glo™ reagent was added to terminate the reaction and deplete the remaining ATP. Then, the kinase detection reagent was 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 was as follows. 4.5 μL of a working solution was added to a 384-well plate. The compound was diluted at 1:3 in succession in DMSO. 0.5 μL of diluted compound solution was added to a 384-well white Optiplate assay plate. The plates were incubated at room temperature for 15 minutes. 5 μL of substrate working solution was added to each well to initiate the reaction. A final ACC reaction solution consisted of: 0.5 nM ACC, 10 μM ATP, 5 μM acetyl-CoA, and 15 mM NaHCO3; and the final concentrations of the compound were measured as follows: 1 μM, 0.333 μM, 0.111 μM, 0.037 μM, 0.0123 μM, 0.00411 μM, 0.00137 μM, 0.000457 μM, 0.000152 μM, and 0.000051 μM. The plates were incubated at room temperature for 30 minutes. 10 μL of ADP-Glo™ reagent was added, and the plates were incubated at room temperature for 40 minutes. 20 μL of kinase detection reagent was added. The plates were incubated at room temperature for 40 minutes, then read on a Perkin Elmer EnVision 2104 plate reader for luminescence in relative light unit (RLU). |
| Affinity data for this assay | |
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