Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon

Abstract

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.

Delineating the role of cooperativity in the design of potent PROTACs for BTK

“…S5). Based on previous reports, affinities of pomalidomide/lenalidomide for CRBN span a range of <1 to >10 μM and highlight that care must be taken when comparing values measured under different conditions (37,38). To minimize variation, we 3B and SI Appendix, Fig.…”

Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders

“…56 The previously reported BRD4 degrader dBET1 was capable of binding to murine CRBN and performed as an active PROTAC in murine cells. 57,58 To test whether our CRBN-based degrader 11 maintains CDK6 degradation across different species, we performed western blotting experiments with the murine myeloid 32D cell line and the murine pro-B-cell line Ba/F3 ( Fig. 2A).…”

Role of cereblon in angiogenesis and in mediating the antiangiogenic activity of immunomodulatory drugs

“…Endothelial cell lines are notoriously difficult to transfect. 21 In order to confirm knockdown could be achieved robustly, we assessed previously used siRNAs for CRBN (Stealth, Invitrogen) in two endothelial cell lines (HUVEC and HMVEC-L), and optimized a commercial (Qiagen flexitube) siRNA for each cell line. CRBN siRNA (Qiagen si3 and Invitrogen Stealth siRNA) reduced CRBN mRNA expression in HUVECs to 14.39% and 12.96% (>85% knockdown efficiency), respectively (P < .0001).…”

“…Both siRNA #3 and siRNA #7 were transfected with DharmaFect (Thermo Fisher Scientific, Walkersville, MD), using a method optimized for endothelial cells and small volume procedures. 21 For all western blot experiments, and a subset of complimentary qPCR experiments, cells were transfected for 24 or 48 hours using CRBN siRNA-STEALTH-1 (Invitrogen) (5 µM) diluted in Lipofectamine 3000 transfection reagent (final concentration, 25 nM) according to the manufacturer's specifications. Unless otherwise stated, experiments are performed in HUVEC cells, transfected with 25 nM Qiagen siCRBN #3, for 24 hours.…”