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Thalidomide and its derivatives, lenalidomide and pomalidomide, are immune modulatory drugs (IMiDs) used in the treatment of haematologic malignancies1,2. IMiDs bind CRBN, the substrate receptor of the CUL4-RBX1-DDB1-CRBN (also known as CRL4CRBN) E3 ubiquitin ligase3, and inhibit ubiquitination of endogenous CRL4CRBN substrates4. Unexpectedly, IMiDs also repurpose the ligase to target new proteins for degradation. Lenalidomide induces degradation of the lymphoid transcription factors Ikaros and Aiolos (also known as IKZF1 and IKZF3)5-7, and casein kinase 1α (CK1α)8, which contributes to its clinical efficacy in the treatment of multiple myeloma5 and 5q-deletion associated myelodysplastic syndrome (del(5q) MDS)8, respectively. How lenalidomide alters the specificity of the ligase to degrade these proteins remains elusive. Here we present the 2.45 Å crystal structure of DDB1-CRBN bound to lenalidomide and CK1α. CRBN and lenalidomide jointly provide the binding interface for a CK1α β-hairpin-loop located in the kinase N-lobe. We show that CK1α binding to CRL4CRBN is strictly dependent on the presence of an IMiD. Binding of IKZF1 to CRBN similarly requires the compound and both, IKZF1 and CK1α, use a related binding mode. Our study provides a mechanistic explanation for the selective efficacy of lenalidomide in del(5q) MDS therapy8,9. We anticipate that high-affinity protein-protein interactions induced by small molecules will provide opportunities for drug development, particularly for targeted protein degradation.
Thalidomide, lenalidomide and pomalidomide are used in the treatment of multiple myeloma1,2. In del(5q) MDS patients, lenalidomide shows pronounced clinical efficacy compared to thalidomide and pomalidomide2,9,10, which has been linked to induced degradation of CK1α (ref. 8). Del(5q) MDS is a haematologic disorder in which deletion of the long arm of chromosome 5 eliminates one casein kinase 1 alpha allele (CSNK1A1). Heterozygous deletion of CSNK1A1 causes haematopoietic stem cell hyperproliferation, whereas homozygous loss of both alleles results in stem cell failure and apoptosis11. CSNK1A1 haploinsufficiency thus sensitizes malignant del(5q) MDS cells to lenalidomide-induced CK1α degradation, while wild-type cells remain unaffected8.
To characterize the binding of CK1α to CRL4CRBN in vitro, we reconstituted the complex and measured the affinity of neddylated CRL4CRBN (N8CRL4CRBN) to CK1α using time-resolved fluorescence resonance energy transfer (TR-FRET) (Extended Data Fig. 1a, b; see Methods). At saturating lenalidomide concentrations (Extended Data Fig. 1c), N8CRL4CRBN and CK1α tightly associate with an apparent dissociation constant...