| Assay Method Information | |
| | Binding Activity of the Compounds Disclosed by the Invention to E3 Ubiquitin Ligase CRBN |
| Description: | The detection is based on homogeneous time-resolved fluorescence (HTRF) technology. When the donor and the acceptor are close, the donor can transfer energy to the acceptor, exciting it to emit light at 665 nm.The donor used in this kit was Europium-labeled GST antibody (GST Eu Cryptate Antibody), and the acceptor was thalidomide labeled with XL665 (Thalidomide-Red reagent). The donor binded to the CRBN protein containing the GST tag. The disclosed compounds in the present, along with lenalidomide and pomalidomide, competitively binded to the CRBN protein with thalidomide. The binding activity of the compounds was determined based on the fluorescence value emitted at 665 nm. The stronger the binding affinity of the compounds, the weaker the signal.The donor and acceptor were diluted 50 times using the binding buffer from the kit (PROTAC binding buffer 1). The CRBN protein was diluted 45 times. The disclosed compound solution at 8 mM was diluted 10 times with the diluent from the kit (1× diluent), followed by a five-fold gradient dilution, performed 7 times sequentially.5 μl of compound, 5 μl of diluted CRBN protein and 10 μl of donor-receptor mixture in equal volume were sequentially added to wells of a white 384-well plate (PE, Part number: 6008280). Incubate at room temperature for 3 hours.The ratio of the emission signals from the acceptor and donor in each individual well is calculated. |
| Affinity data for this assay | |
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