Fig 2: DDB1-based androgen receptor PROTACs.(A) Structures of MM-02–057-based AR PROTACs with different linker lengths. (B) AR levels in LNCaP prostate cancer cells. LNCaP cells were treated with DMSO vehicle or PROTACs for 24 h after which AR and loading control GAPDH levels were assessed by Western blotting. (C) TMT-based quantitative proteomic profiling of MM-03–75 in LNCaP cells. LNCaP cells were treated with MM-03–75 (10 µM) for 24 h. Red points denote statistically significant downregulated proteins including AR. Blots and proteomic data are representative of n=3 biologically independent replicates per group.

Fig 3: DDB1-dependence of BRD4 degradation.(A) Attenuation of MM-02–08-mediated BRD4 degradation with proteasome and NEDDylation inhibitors. HEK293T cells were treated with DMSO or BTZ (1 µM) or MLN4924 (1 µM) for 1 h prior to MM-02–08 (1 µM) treatment for 24 hours. BRD4 and loading control GAPDH were assessed by Western blotting. All blots are representative of n=3 biologically independent replicates/group. (B) DDB1 knockdown attenuates MM-02–08-mediated BRD4 degradation. Stable short-hairpin mediated control and DDB1 knockdown HEK293T cells were treated with DMSO vehicle or MM-02–08 (1 µM) treatment for 24 hours and BRD4, DDB1, and loading control actin were assessed by Western blotting. (C) Reconstitution of various components of the CUL4A complex with ubiquitin and BRD4 treated with DMSO vehicle or MM-02–08 (1 µM) treatment for 1 hour. FLAG-BRD4 levels, including higher molecular weight ubiquitinated FLAG-BRD4 levels were assessed by Western blotting. All blots are representative of n=3 biologically independent replicates/group.

Fig 4: Characterization of DDB1 covalent recruiter MM-02–57.(A) Structure of DDB1 covalent ligand hit MM-02–57. (B) Gel-based ABPP of MM-02–57 against rhodamine-functionalized cysteine-reactive iodoacetamide probe (IA-rhodamine) labeling of pure DDB1 protein. (C) Gel-based ABPP of MM-02–57 against IA-rhodamine labeling of pure RNF114. (D) Structure of alkyne-functionalized probe MM-02–54. (E) Gel-based ABPP of MM-02–57 against IA-rhodamine labeling of pure DDB1 protein. For gels in (B, C, E), DDB1 or RNF114 was pre-incubated with DMSO or the covalent ligand for 30 min prior to IA-rhodamine labeling (100 nM for (B, E) and 10 µM for (C)) for 30 min after which proteins were resolved by SDS/PAGE and IA-rhodamine labeling was visualized by in-gel fluorescence and loading was assessed by silver staining. (F) Gel-based ABPP of MM-02–57 against MM-02–54 labeling of pure DDB1 protein. Probe-labeled proteins were subjected to CuAAC-mediated appendage of a rhodamine-azide after which proteins were resolved on SDS/PAGE and probe labeling was assessed by in-gel fluorescence and loading was assessed by silver staining. (G) LC-MS/MS analysis of MM-02–57 modification on DDB1. DDB1 was labeled with MM-02–57 (50 µM) for 30 min and subsequently tryptically digested for LC-MS/MS analysis. Shown are the MS/MS data for the MM-02–57-modified C173-containing DDB1 peptide. (H) Reconstituted CUL4A ubiquitination assay with MM-02–057. Various components of the complex were pre-incubated with DMSO vehicle or MM-02–57 (50 µM) for 30 min, after which FLAG-Ubiquitinated proteins were detected by Western blotting. Gels shown in (B, C, E, F, H) are representative gels from n=3 biologically independent replicates/group.

Fig 5: DDB1 target engagement and selectivity of MM-02–057 in cells.(A) MM-02–54 target engagement of DDB1 in cells. HEK293T cells were treated with DMSO vehicle or MM-02–54 (xxx µM) for xxx hours, after which resulting cell lysates were subjected to CuAAC-mediated appendage of an azide-functionalized biotin handle onto probe-labeled proteins. Probe-labeled proteins were avidin-enriched, eluted, and resolved on SDS/PAGE and DDB1 and an unrelated protein GAPDH input and pulldown were detected by Western blotting. (B) IsoDTB-ABPP analysis of MM-02–57 in HEK293T cells. HEK293T cells were treated with DMSO vehicle or MM-02–57 (50 µM) for 4 hours after which resulting control and treated lysates were labeled with an alkyne-functionalized iodoacetamide probe (IA-alkyne) for 1 h. Probe-labeled proteins were subjected to CuAAC to append either an isotopically light (for control) or heavy (for treated) azide-functionalized desthiobiotin handle, after which probe-labeled proteins were avidin-enriched, digested with trypsin, and probe-modified peptides were eluted and analyzed by LC-MS/MS. Control/treated probe-modified peptides were quantified and plotted. The points shown in red were targets that showed ratios of >8 with adjusted p-values <0.05. The full dataset can be found in Table S2. Data in (A, B) are from n=3 biologically independent replicates per group.