Extended Data Figure 4. Detailed analysis of CRBN-compound interactions.

(a) Thermal denaturation assays of thalidomide binding to hsDDB1-hsCRBN wild type (CRBN wt) and to an hsDDB1-hsCRBN mutant harbouring mutations Tyr386Ala and Trp388Ala (CRBN^YW/AA). While the mutant showed no sign of binding to thalidomide, wild type CRBN caused a shift in the thermal denaturation curve indicative of compound binding. (b) As in (a), but using lenalidomide or (c) pomalidomide. (d) Increasing amounts of hsDDB1-hsCRBN were mixed with 20 nM Cy5-coupled thalidomide (compound 5). Protein-compound interactions were quantified using fluorescence polarisation of the Cy5 dye. Curve fitting to a model assuming one binding site resulted in a K_D of 121.6 ± 23.2 nM. Data shown are the means ± SD (n=3).(e) Competitive titration of thalidomide to hsDDB1-hsCRBN (50 nM) and Cy5-thalidomide (20 nM); EC₅₀ values were used to calculate a K_i⁵⁰ (equivalent to the K_D of thalidomide to hsDDB1-hsCRBN) of 249.20 nM. Similar titrations were carried out for (f) lenalidomide and (g) pomalidomide resulting in K_i values of 177.80 nM and 156.60 nM, respectively. For all competitive titrations (e–g) data shown are the means ± SD (n=3).(h) Affinities of thalidomide analogues for the CRBN receptor.