ABSTRACT
Targeted protein degradation via small-molecule modulation of cereblon offers vast potential for the development of new therapeutics. Cereblon-binding therapeutics carry the safety risks of thalidomide, which caused an epidemic of severe birth defects characterized by forelimb shortening or phocomelia. Here we show that thalidomide is not teratogenic in transgenic mice expressing human cereblon, indicating that binding to cereblon is not sufficient to cause birth defects. Instead, we identify SALL4 as a thalidomide-dependent cereblon neosubstrate. Human mutations in SALL4 cause Duane-radial ray, IVIC, and acro-renal-ocular syndromes with overlapping clinical presentations to thalidomide embryopathy, including phocomelia. SALL4 is degraded in rabbits but not in resistant organisms such as mice because of SALL4 sequence variations. This work expands the scope of cereblon neosubstrate activity within the formerly 'undruggable' C2H2 zinc finger family and offers a path toward safer therapeutics through an improved understanding of the molecular basis of thalidomide-induced teratogenicity.
Subject(s)
Gene Expression Regulation , Peptide Hydrolases/chemistry , Teratogens/chemistry , Thalidomide/chemistry , Transcription Factors/chemistry , Adaptor Proteins, Signal Transducing , Animals , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Homozygote , Humans , Immunohistochemistry , Induced Pluripotent Stem Cells , Ligands , Male , Mice , Mice, Transgenic , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Peptide Hydrolases/genetics , Proteolysis , Rabbits , Testis/metabolism , Transcription Factors/genetics , Ubiquitin-Protein Ligases/metabolism , Zinc FingersABSTRACT
The drugs lenalidomide and pomalidomide bind to the protein cereblon, directing the CRL4-CRBN E3 ligase toward the transcription factors Ikaros and Aiolos to cause their ubiquitination and degradation. Here we describe CC-220 (compound 6), a cereblon modulator in clinical development for systemic lupus erythematosis and relapsed/refractory multiple myeloma. Compound 6 binds cereblon with a higher affinity than lenalidomide or pomalidomide. Consistent with this, the cellular degradation of Ikaros and Aiolos is more potent and the extent of substrate depletion is greater. The crystal structure of cereblon in complex with DDB1 and compound 6 reveals that the increase in potency correlates with increased contacts between compound 6 and cereblon away from the modeled binding site for Ikaros/Aiolos. These results describe a new cereblon modulator which achieves greater substrate degradation via tighter binding to the cereblon E3 ligase and provides an example of the effect of E3 ligase binding affinity with relevance to other drug discovery efforts in targeted protein degradation.
Subject(s)
Heterocyclic Compounds, 4 or More Rings/pharmacology , Ikaros Transcription Factor/metabolism , Peptide Hydrolases/chemistry , Peptide Hydrolases/metabolism , Proteolysis/drug effects , Adaptor Proteins, Signal Transducing , Cell Line, Tumor , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Fluorescence Resonance Energy Transfer , Heterocyclic Compounds, 4 or More Rings/chemistry , Heterocyclic Compounds, 4 or More Rings/metabolism , Humans , Lenalidomide/chemistry , Lenalidomide/metabolism , Morpholines , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Phthalimides , Piperidones , Protein Binding , Ubiquitin-Protein LigasesABSTRACT
Immunomodulatory drugs bind to cereblon (CRBN) to confer differentiated substrate specificity on the CRL4(CRBN) E3 ubiquitin ligase. Here we report the identification of a new cereblon modulator, CC-885, with potent anti-tumour activity. The anti-tumour activity of CC-885 is mediated through the cereblon-dependent ubiquitination and degradation of the translation termination factor GSPT1. Patient-derived acute myeloid leukaemia tumour cells exhibit high sensitivity to CC-885, indicating the clinical potential of this mechanism. Crystallographic studies of the CRBN-DDB1-CC-885-GSPT1 complex reveal that GSPT1 binds to cereblon through a surface turn containing a glycine residue at a key position, interacting with both CC-885 and a 'hotspot' on the cereblon surface. Although GSPT1 possesses no obvious structural, sequence or functional homology to previously known cereblon substrates, mutational analysis and modelling indicate that the cereblon substrate Ikaros uses a similar structural feature to bind cereblon, suggesting a common motif for substrate recruitment. These findings define a structural degron underlying cereblon 'neosubstrate' selectivity, and identify an anti-tumour target rendered druggable by cereblon modulation.
Subject(s)
Antineoplastic Agents/pharmacology , Peptide Hydrolases/metabolism , Peptide Termination Factors/metabolism , Phenylurea Compounds/pharmacology , Thalidomide/analogs & derivatives , Adaptor Proteins, Signal Transducing , Amino Acid Motifs , Antineoplastic Agents/chemistry , Binding Sites , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Humans , Ikaros Transcription Factor/chemistry , Ikaros Transcription Factor/metabolism , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Peptide Hydrolases/chemistry , Peptide Termination Factors/chemistry , Peptide Termination Factors/deficiency , Phenylurea Compounds/chemistry , Protein Binding , Proteolysis/drug effects , Substrate Specificity , Thalidomide/chemistry , Thalidomide/pharmacology , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolismABSTRACT
The Cul4-Rbx1-DDB1-Cereblon E3 ubiquitin ligase complex is the target of thalidomide, lenalidomide and pomalidomide, therapeutically important drugs for multiple myeloma and other B-cell malignancies. These drugs directly bind Cereblon (CRBN) and promote the recruitment of substrates Ikaros (IKZF1) and Aiolos (IKZF3) to the E3 complex, thus leading to substrate ubiquitination and degradation. Here we present the crystal structure of human CRBN bound to DDB1 and the drug lenalidomide. A hydrophobic pocket in the thalidomide-binding domain (TBD) of CRBN accommodates the glutarimide moiety of lenalidomide, whereas the isoindolinone ring is exposed to solvent. We also solved the structures of the mouse TBD in the apo state and with thalidomide or pomalidomide. Site-directed mutagenesis in lentiviral-expression myeloma models showed that key drug-binding residues are critical for antiproliferative effects.
Subject(s)
Angiogenesis Inhibitors/pharmacology , DNA-Binding Proteins/metabolism , Peptide Hydrolases/metabolism , Thalidomide/analogs & derivatives , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Angiogenesis Inhibitors/chemistry , Animals , Crystallography, X-Ray , DNA-Binding Proteins/chemistry , Humans , Lenalidomide , Mice , Molecular Docking Simulation , Molecular Sequence Data , Peptide Hydrolases/chemistry , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Sequence Alignment , Thalidomide/chemistry , Thalidomide/pharmacology , Ubiquitin-Protein LigasesABSTRACT
Localized induction of bone formation is essential during orthopedic procedures that involve skeletal repair, such as surgical treatment of non-union bone fractures and degenerative disk disease. Herein we disclose the synthesis and biological evaluation of novel oxysterol derivatives designed as anabolic bone growth agents. Structure-activity relationship studies of oxysterol 4 have identified analogues such as 18, 21 and 30. These new analogues are characterized by higher potency in an osteoblast differentiation assay and/or by increased metabolic stability in human liver microsomes. Oxysterols 4, 18 and 21 were evaluated in vivo in a rat spinal fusion model.
