| Assay Method Information | |
| | Enzyme Level Activity Assay |
| Description: | In the present disclosure, the enzyme level activity the compounds is detected by using the MDM2/p53 protein binding experiment and using a TR-FRET method. The steps were specifically as follows: an Echo pipette (Labcyte) was used to perform a 3.162-fold serial dilution on the test compounds, and each compound was diluted in 11 concentrations and 250 nL for each concentration was transferred to a 384-well plate, with two replicate wells set for each compound concentration. The well with positive compound (100% inhibition) added was set as a positive control, and the well with DMSO only as a negative control. The GST-MDM2 protein (R & D-E3-202-050) was diluted to 0.625 nM with a buffer (125 mM NaCl, 1 mM DTT, 0.01% Gelatin (animal gelatin), 0.1% Pluronic f-127 (polyether), 1 PBS), and 20 μL of the diluent was added to the 384-well plate. After centrifugation and shaking, the 384-well plate was placed in a 23° C. incubator and incubated for 20 min. The His-p53 protein (R & D-SP-450-020) was diluted to 12.5 nM with a buffer, and 20 μL of the diluent was added to the 384-well plate. After centrifugation and shaking, the 384-well plate was placed in a 23° C. incubator and incubated for 60 min. The Eu2+ anti-GST antibody (Cisbio-61GSTKLB) and XL665 anti-His antibody (Cisbio-61HISXLB) were diluted with a buffer, and the diluted mixture contains 0.3 nM Eu2+ anti-GST antibody and 9 nM XL665 anti-His antibody. 10 μL of the mixture of the two antibodies was added to the 384-well plate. After centrifugation and shaking, the 384-well plate was placed in a 23° C. incubator and incubated for 20 h. Reading was performed on an Envision multifunctional microplate reader (PerkinElmer) (excitation light at 340 nm, and emission lights at 665 nm and 615 nm). Ratio=signal intensity at 665 nm/signal intensity at 615 nm×10000 is used to calculate the inhibition ratio, and the formula is as follows: inhibition ratio=(Ratio of the well with compounds added−Ratio of the negative control)/(Ratio of the positive control−Ratio of the negative control)*100%, and the IC50. |
| Affinity data for this assay | |
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