| Assay Method Information | |
| | CRBN-DDB1 ligand-displacement AlphaScreen Assay |
| Description: | A thalidomide competition AlphaScreen assay was employed to measure the binding affinity (IC50) of thalidomide conjugates and novel IMiDs to CRBN-DDB1. In 384-well AlphaPlates (Perkin Elmer), 50 nM CRBN-DDB1 and 125 nM biotin-thalidomide were diluted in 20 uL assay buffer (50 mM HEPES pH 7.4, 200 mM NaCl, 1 mM TCEP, and 0.1% BSA) containing competitor compound or DMSO. Following a 30 min incubation, 20 uL detection solution containing Streptavidin Donor Beads and Nickel Chelate AlphaLISA Acceptor Beads diluted to 20 ng/uL in assay buffer was added to each well. After 1 hr incubation at RT, luminescence was measured on the Envision 2104 plate reader. Percent activity values were calculated by setting the average background (no protein wells) to 0% the average DMSO wells to 100% activity. Standard deviations were determined from at least four replicate measurements for compound concentration. Data were analyzed and plotted using GraphPad PRISM v6 and CRBN Alpha IC50 values were determined using the log(inhibitor) vs normalized response−variable slope analysis module. |
| Affinity data for this assay | |
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