Design of a Cereblon construct for crystallographic and biophysical studies of protein degraders

• Published in: bioRxiv
• Main Authors: Kroupova, Alena, Spiteri, Valentina A, Furihata, Hirotake, Darren, Darren, Ramachandran, Sarath, Rutter, Zoe, Chakraborti, Sohini, Haubrich, Kevin, Pethe, Julie, Gonzales, Denzel, Wijaya, Andre, Rodriguez-Rios, Maria, Lynch, Dylan M, Farnaby, William, Nakasone, Mark, Zollman, David, Ciulli, Alessio
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 20.01.2024
• Subjects: Proteins; Thalidomide; Ubiquitin-protein ligase
• DOI: 10.1101/2024.01.17.575503

The ubiquitin E3 ligase cereblon (CRBN) is the target of therapeutic drugs thalidomide and lenalidomide and is recruited by most targeted protein degraders (PROTACs and molecular glues) in clinical development. Biophysical and structural investigation of CRBN has been limited by current constructs that either require co-expression with the adaptor DDB1 or inadequately represent full-length protein, with high-resolution structures of degraders ternary complexes remaining rare. We present the design of CRBNmidi, a construct that readily expresses from E. coli with high yields as soluble, stable protein without DDB1. We benchmark CRBNmidi for wild-type functionality through a suite of biophysical techniques and solve high-resolution co-crystal structures of its binary and ternary complexes with degraders. We qualify CRBNmidi as an enabling tool to accelerate structure-based discovery of the next generation of CRBN based therapeutics.Competing Interest StatementA.C. is a scientific founder, shareholder and advisor of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms. The Ciulli laboratory receives or has received sponsored research support from Almirall, Amgen, Amphista Therapeutics, Boehringer Ingelheim, Eisai, Merck KaaG, Nurix Therapeutics, Ono Pharmaceutical and Tocris-Biotechne.