| Assay Method Information | |
| | In Vitro Enzymatic Inhibition Activity Assay |
| Description: | Enzymatic activity assays of compounds of this patent against MAT2A (to detect half inhibitory concentration IC50 values of the compounds) were performed using the phosphate detection kit PiColorLockTM (ab270004, purchased from Abcam). This method utilized the principle that purified human MAT2A enzyme catalyzes the formation of S-adenosylmethionine (SAM) from L-methionine and ATP to release free phosphate, and used the spectrophotometric method to quantify phosphate, so as to determine the enzymatic activity of MAT2A. Compounds were diluted in a 10-fold gradient starting at 1 mM with 100% DMSO (for a total of 8 concentrations) and 2 μL of each concentration was added to 48 μL of reaction buffer (50 mM Tris-HCl PH 7.5, 50 mM KCl, 10 mM MgCl2, 0.01% Brij-35, 0.01% BSA, 1 mM DTT) and mixed well. The final diluted compound was prepared, and 5 μL of MAT2A enzyme (final concentration of 10 nM), 5 μL of ATP (final concentration of 50 μM), and 5 μL of L-methionine (final concentration of 100 μM) were added to each well of a 384-well plate (#3701, purchased from CORNING). After the 384-well plate was placed in the incubator at 23° C. for 6 h, 5 μL of PiColorLock reagent diluted Accelerator (dilution ratio 1:100) was added to each well, and the reaction was stopped by incubating in the incubator at 23° C. for 5 min. 2 μL of Stabiliser was added, the absorbance was read at 630 nm on BMG CLARIOstar, and the IC50 value of the compound was calculated by using GraphPad Prism software. |
| Affinity data for this assay | |
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