Development of Substituted Phenyl Dihydrouracil as the Novel Achiral Cereblon Ligands for Targeted Protein Degradation.

Glutarimides such as thalidomide, pomalidomide, and lenalidomide are the most frequently used ligands to recruit E3 ubiquitin ligase cereblon (CRBN) for the development of proteolysis-targeting chimeras (PROTACs). Due to the rapid and spontaneous racemization of glutarimides, most CRBN-recruiting PROTACs are synthesized as a mixture of racemates or diastereomers. Since the (S)-enantiomer is primarily responsible for binding to CRBN, the existence of the largely inactive (R)-enantiomer complicates the drug development process. Herein, we report that substituted achiral phenyl dihydrouracil (PDHU) can be used as a novel class of CRBN ligands for the development of PROTACs. Although the parent PDHU has a minimal binding affinity to CRBN, we found that some substituted PDHUs had a comparable binding affinity to lenalidomide. Structural modeling provided a further understanding of the molecular interactions between PDHU ligands and CRBN. PDHUs also have greater stability than lenalidomide. Finally, potent BRD4 degraders were developed by employing trisubstituted PDHUs.

The efficacy of combination treatment with elotuzumab and lenalidomide is dependent on crosstalk between natural killer cells, monocytes and myeloma cells.

Patients with refractory relapsed multiple myeloma respond to combination treatment with elotuzumab and lenalidomide. The mechanisms underlying this observation are not fully understood. Furthermore, biomarkers predictive of response have not been identified to date. To address these issues, we used a humanized myeloma mouse model and adoptive transfer of human natural killer (NK) cells to show that elotuzumab and lenalidomide treatment controlled myeloma growth, and this was mediated through CD16 on NK cells. In co-culture studies, we showed that peripheral blood mononuclear cells from a subset of patients with refractory relapsed multiple myeloma were effective killers of OPM2 myeloma cells when treated with elotuzumab and lenalidomide, and this was associated with significantly increased expression of CD54 on OPM2 cells. Furthermore, elotuzumab- and lenalidomide-induced OPM2 cell killing and increased OPM2 CD54 expression were dependent on both monocytes and NK cells, and these effects were not mediated by soluble factors alone. At the transcript level, elotuzumab and lenalidomide treatment significantly increased OPM2 myeloma cell expression of genes for trafficking and adhesion molecules, NK cell activation ligands and antigen presentation molecules. In conclusion, our findings suggest that multiple myeloma patients require elotuzumab- and lenalidomide-mediated upregulation of CD54 on autologous myeloma cells, in combination with NK cells and monocytes to mediate an effective anti-tumor response. Furthermore, our data suggest that increased myeloma cell CD54 expression levels could be a powerful predictive biomarker for response to elotuzumab and lenalidomide treatment.

Discovery of novel BCR-ABL PROTACs based on the cereblon E3 ligase design, synthesis, and biological evaluation.

Protein degradation is a promising strategy for drug development. Proteolysis-targeting chimeras (PROTACs) hijacking the E3 ligase cereblon (CRBN) exhibit enormous potential and universal degradation performance due to the small molecular weight of CRBN ligands. In this study, the CRBN-recruiting PROTACs were explored on the degradation of oncogenic fusion protein BCR-ABL, which drives the pathogenesis of chronic myeloid leukemia (CML). A series of novel PROTACs were synthesized by conjugating BCR-ABL inhibitor dasatinib to the CRBN ligand including pomalidomide and lenalidomide, and the extensive structure-activity relationship (SAR) studies were performed focusing on optimization of linker parameters. Therein, we uncovered that pomalidomide-based degrader 17 (SIAIS056), possessing sulfur-substituted carbon chain linker, exhibits the most potent degradative activity in vitro and favorable pharmacokinetics in vivo. Besides, degrader 17 also degrades a variety of clinically relevant resistance-conferring mutations of BCR-ABL. Furthermore, degrader 17 induces significant tumor regression against K562 xenograft tumors. Our study indicates that 17 as an efficacious BCR-ABL degrader warrants intensive investigation for the future treatment of BCR-ABL+ leukemia.

A Cell-Based Target Engagement Assay for the Identification of Cereblon E3 Ubiquitin Ligase Ligands and Their Application in HDAC6 Degraders.

Proteolysis-targeting chimeras (PROTACs) is a paradigm shift for small-molecule drug discovery. However, limited E3 ubiquitin ligase ligands with cellular activity are available. In vitro binding assays involve the expression and purification of a large amount of proteins and they often yield ligands that are inactive in cell-based assays due to poor cell permeability, stability, and other reasons. Herein, we report the development of a practical and efficient cell-based target engagement assay to evaluate the binding affinity of a small library of cereblon ligands to its E3 ligase in cells. Selected cell-permeable E3 ligase ligands derived from this assay are then used to construct HDAC6 degraders with cellular protein degradation activity. Because the assay does not involve any genetic engineering, it is relatively easy to transfer from one cell type to a different one.

A Cereblon Modulator (CC-220) with Improved Degradation of Ikaros and Aiolos.

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.