| Assay Method Information | |
| | etermination of IC50 of Inhibition of Enzymatic Activity of ACC1 and ACC2 by the Compound of the Present Disclosure |
| Description: | The degree of inhibition of the enzymatic activity of recombinant human ACC1, ACC2 proteins under in-vitro conditions by the preferred compounds of the present disclosure is determined by the following method.The principle of the method was based on the reaction of ACC protein-catalyzed acetyl-CoA to form malonyl-CoA. ATP was consumed during this reaction and ADP was produced. The produced ADP was reconverted into ATP by using ADP-Glo Kinase Kit from Promega. This part of ATP reacted with the luciferase-fluorescence in the kit, and generated chemiluminescent signals. Thus, by measuring the intensity of chemiluminescent signal, the amount of ADP produced in the catalytic reaction was reflected, thereby indirectly determining the enzymatic activity of ACC protein and the effect of the tested compound on the enzymatic activity. The main reagents used included ACC1, ACC2 proteins (purchased from BPS bioscience, ACC1 Art. No. 50200, ACC2 Art. No. 50201), acetyl-CoA (acetyl-CoA, purchased from Sigma, Art. No. A2056), NaHCO3 (purchased from Sigma, Art. No. S6014), ADP-Glo Kinase assay kit (purchased from Promega, Art. No. V9102).The test procedure was briefly described as follows: firstly, a 1× buffer solution required for the reaction was prepared, comprising: 50 mM HEPES (pH7.4 purchased from Invitrogen, Art. No. 15630), 2 mM magnesium chloride (MgCl2, purchased from Sigma, Art. No. M1028), 2 mM potassium citrate (Potassium citrate, purchased from Sigma, Art. No. 89306), 0.01% Brij-35 detergent (purchased from Merck, Art. No. 203728), 2 mM DTT (purchased from Sigma, Art. No. D0632). The test compound powder was dissolved in DMSO to prepare a stock solution having a concentration of 10 mM, followed by three-fold dilution to prepare the concentration required for the test, and each compound was set at 10 concentration points ranging from 10 μm to 0.5 nM. Firstly, appropriate amount of ACC protein (2 nM) was added to a 384-well microplate, and then diluted test compound solutions were added to each well. A duplicate well was provided at each concentration, and a solution control group (blank group) and a negative control group (DMSO group) were set at the same time. The 384-well plates were then shaken on a microwell plate oscillator and incubated for 15 minutes at room temperature. Thereafter, a substrate mixture containing ATP, acetyl-CoA and NaHCO3 which was diluted with the aforementioned buffer solution was added to each well to start the reaction, and the final concentrations of the three components were respectively ATP 20 μM, acetyl-CoA 10 μM, and NaHCO3 30 mM. After reacting for 30 minutes at room temperature, the corresponding reaction solution and detection solution were added to each well according to the method in the specification of ADP-Glo Kinase assay kit (referring to the specification of the kit for specific methods). Finally, the relative light unit (RLU) values of each well were tested using an Envision 2104 multi-function microplate reader (Perkin Elmer). The percentage inhibition rate of a compound at a concentration on ACC enzyme activity was calculated by the following formula:Inhibition rate %=[(RLU average value of negative control wells−RLU average value of blank control wells)−(RLU average values of test wells−RLU average values of blank control wells)]/(RLU average values of negative control wells−RLU average values of blank control wells)*100Finally, nonlinear regression analysis of the logarithm of the concentration of the compound and the percentage inhibition rate of the corresponding concentration was carried out in the GraphPad Prism5 software to obtain the half maximal inhibitory concentration (IC50) of the compound. |
| Affinity data for this assay | |
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