Fig. 2: Biochemical and cell-based analyses of 6-position-modified lenalidomides.
From: Lenalidomide derivatives and proteolysis-targeting chimeras for controlling neosubstrate degradation
a Chemical structures of thalidomide (Th), pomalidomide (Po), lenalidomide (Le), 6-fluoro lenalidomide (F-Le), 6-chloro lenalidomide (Cl-Le) and 6-bromo lenalidomide (Br-Le). b Dose-dependent interaction assay using AlphaScreen (AS) technology. The CRBN–IKZF1, CRBN–SALL4, and CRBN–PLZF complex formation was analysed. The relative AS signals are expressed as the luminescence signal relative to the luminescence signal of dimethylsulfoxide (DMSO), which is considered 1. Error bars denote standard deviations (independent experiments, n = 3). c In vitro ubiquitination assay of SALL4 and IKZF1 by CRL4CRBN. Purified FLAG-GST-IKZF1 or -SALL4 were mixed with recombinant CRL4CRBN, E1, E2, and HA-Ub, and ubiquitination reactions were performed in the presence of DMSO, 20-µM pomalidomide (Po), 20-µM Le, 20-µM F-Le, 20-µM Cl-Le, or 20-µM Br-Le. Ubiquitinated SALL4 and IKZF1 were immunoprecipitated using an anti-FLAG antibody. The experiment was repeated twice independently, with similar results. d In the cell ubiquitination assay of SALL4 and IKZF1 by CRL4CRBN. HEK293T cells were transfected with pcDNA3-HA-ubiquitin and pcDNA3.1-FLAG-CRBN and pcDNA3.1-AGIA-SALL4 or -IKZF1 and treated with DMSO, 1-µM Po, 10-µM Le, 10-µM F-Le, 10-µM Cl-Le, or 10-µM Br-Le in the presence of 10-µM MG132 for 8 h. Ubiquitinated SALL4 and IKZF1 were immunoprecipitated using an anti-AGIA antibody. The experiment was repeated twice independently, with similar results. e–g Immunoblot analysis of dose-dependent neosubstrate degradation in (e) MM1.S, (f) HuH7, or (g) NTERA-2 cells. Each cell line was treated with DMSO, Po, Le, F-Le, Cl-Le, or Br-Le for 24 h. The experiment was independently repeated thrice, with similar results. Source data are provided as a Source data file.
