Development of Phenyl-substituted Isoindolinone- and Benzimidazole-type Cereblon Ligands for Targeted Protein Degradation

Abstract

Thalidomide, pomalidomide and lenalidomide, collectively referred to as immunomodulatory imide drugs (IMiDs), are frequently employed in proteolysis-targeting chimeras (PROTACs) as cereblon (CRBN) E3 ligase-recruiting ligands. However, their molecular glue properties that co-opt the CRL4^CRBN to degrade its non-natural substrates may lead to undesired off-target effects for the IMiD-based PROTAC degraders. Herein, we reported a small library of potent and cell-permeable CRBN ligands, which exert high selectivity over the well-known CRBN neo-substrates of IMiDs by structure-based design. They were further utilized to construct bromodomain-containing protein 4 (BRD4) degraders, which successfully depleted BRD4 in the tested cells. Overall, we reported a series of functionalized CRBN recruiters that circumvent the promiscuity from traditional IMiDs, and this study is informative to the development of selective CRBN-recruiting PROTACs for many other therapeutic targets.

Keywords: CRBN; IMiDs; PROTAC; Targeted Protein Degradation.

Figure 1.

Relative binding affinities of CRBN ligands to CRBN in cellular and biochemical assays. a) Principle of the cellular target engagement binding assay, and the structure of HDAC6-targeting PROTAC degrader 1. b) Results of the cell-based TE assay. MM.1S cells were pretreated with 3 μM CRBN ligand or DMSO for 1 h, then with 100 nM degrader 1 or DMSO for 5 h, HDAC6 levels are analyzed by in-cell ELISA. Bar graphs were generated as means of HDAC6 relative level (n = 3) with ±SD as the error bar. c) Result of the FP competition assay. 100 nM recombinant DDB1/CRBN protein complex was incubated with 8 nM fluorescent probe (FITC-thalidomide) and 150 nM CRBN binder or DMSO in the assay buffer 50 mM HEPES, 75 mM NaCl, 0.01% Triton X-100, pH 7.4, the total volume is 20 uL. Fluorescence polarization signal was converted to % inhibition by comparing to DMSO control.

Figure 2.

Selectivity of new CRBN ligands and structural modeling of CRBN-pomalidomide/1A complexes. a) Activity of selected CRBN ligands towards CRBN neosubstrate degradation. MM.1S cells were treated with 1 μM CRBN ligands or DMSO for 6 h and degradation of CRBN neosubstrates were examined by immunoblot assay. b) Crystal structure of CRBN-pomalidomide-IKZF1 (PDB 6H0F). C5 and C6 positions on the ligand for potential ring substituent are circled out in green, which are too close to IKZF1. Replacing NH₂ with a ring disrupts the important H-bond network between CRBN E377 and IKZF1 GLN146. c) It is impossible to fit ligand 1A into the pocket without disturbing the known ternary binding pose of CRBN-IKZF1, as further evidenced by docking which only gave poses outside of pocket with poor scores. d) The same docking as in (c) but was performed with pomalidomide, the docked pose fully overlaps with the correct crystal pose, and with a favorable score of −12.4. e) Docking of 1A to CRBN only from a different crystal structure (PDB 4TZ4) with E377 more open gives crystal overlapping poses, and with a favorable score of −10.9, slightly better than the 3 canonical CRBN ligands, consistent with the experimental results.

Figure 3.

BRD4 PROTACs derived from CRBN ligands with a 2,3-dihydro-2-oxo-1H-benzimidazole core. a) Structures of aldehyde intermediates 5X, 6X and BRD4 degraders 5Y, 6Y derived from CRBN ligands with a 2,3-dihydro-2-oxo-1H-benzimidazole core. b) Western blot result of BRD4 degradation induced by BRD4 degraders 5Y and 6Y series. SU-DHL-4 cells were treated with 100 nM or 1 μM degrader or DMSO for 24 h. c) Time course of the most potent BRD4 degrader 5Y-3. SU-DHL-4 cells were treated with 100 nM 5Y-3 and incubated for different times up to 12 h as indicated. d) Mechanism study of 5Y-3. SU-DHL-4 cells were pretreated with 1 μM bortezomib, MG132, MLN4924 or DMSO for 1 h, followed by treatment of 100 nM 5Y-3 for 24 h. e) Dose response of 5Y-3 in SU-DHL-4 and MV-4–11 cells. Cells were treated with 5Y-3 of different concentrations as indicated for 24 h. f) Selectivity test of 1G, 5P, 6P and 5Y-3. SU-DHL-4 cells were treated with 1 μM CRBN ligands or 100 nM 5Y-3 or DMSO for 8 h.

Scheme 1.

Chemical structures of immunomodulatory agents and CRBN ligands bearing isoindolinone or 2,3-dihydro-2-oxo-1H-benzimidazole cores.