Figure 1

Binding assays of deuterium-substituted (S)- and (R)-thalidomides with human CRBN TBD. (a) Chemical structures of deuterated (S)- and (R)-thalidomides, (S)-D-Thal and (R)-D-Thal, respectively. Atom numbering is shown in the (S)-D-Thal chemical structure. The hydrogen atom at the chiral centre C3 atom of the glutarimide moiety is substituted with a deuterium atom. (b) Competitive elution assay using thalidomide-immobilized beads coupled with racemic thalidomide. Beads were mixed with extracts from 293 T cells expressing FLAG-HA-CRBN and washed three times with 0.5% NP-40 lysis buffer and bound proteins were eluted with wash buffer containing 1 mM deuterated (S)- or (R)-thalidomide ((S)-D-Thal or (R)-D-Thal), (S)- or (R)-thalidomide ((S)-Thal or (R)-Thal) or DMSO for the indicated time. The eluate was then analyzed by SDS-PAGE and immunoblotting (IB). (c) Inhibitory effects of thalidomide enantiomers on auto-ubiquitylation of FH-CRBN were detected in the presence of MG132. Cells were treated with DMSO or the indicated concentrations of (S)-D-Thal or (S)-D-Thal for 4 hours prior to harvesting. Full-length blots in (b) and (c) are presented in Supplementary Fig. 8.