Extended Data Fig. 1: SB-405483 binds allosterically to CRBN in vitro.
From: Identification of an allosteric site on the E3 ligase adapter cereblon
(a) Schematic of TR-FRET competition assay. (b) ASMS data demonstrating binding of both orthosteric and allosteric ligands. (c) ASMS signal for SB-405483 over increasing concentrations of SB-405483 and pomalidomide. (d) ASMS signal for pomalidomide over increasing concentrations of SB-405483 and pomalidomide. (e) TR-FRET IC50 values of orthosteric ligands in the presence or absence of 25 µM SB-405483. (f) X-ray maps around the ligand. OMIT difference map (Fo-Fc), after removal of the ligand from the final X-ray structure model, is shown. The map is contoured at +/- 3 sigma, in blue and green respectively. The modelling of the binding mode of ligands are unambiguous with all elements of the molecule clearly defined. (g) Side view of allosteric site. (h) Top view of allosteric binding site. (i) Bottom view of allosteric binding site. (j) 4CI2 overlaid with SB-405483. (k) 4TZ4 overlaid with SB-405483. (l) Crystal structure of human CRBN/DDB1 with SB-405483. (m) Front and rear site view of allosteric binding site from the crystal structure obtained in this study. Residues in grey are 100% conserved across species. Residues in dark red are conserved in 75-99% of species. Residues in pink are conserved in 50-74% of species. (n) AlphaFold structure of Mus musculus CRBN allosteric site, X-ray structure of Gallus gallus CRBN allosteric site (PDB: 4CI2), and AlphaFold structure of Danio rerio CRBN allosteric site with residues that differ from the Homo sapiens CRBN allosteric site in red. ASMS data are the mean values resulting from n = 2 biologically independent samples. TR-FRET data are mean ± s.d. from n = 4 biologically independent samples.
