glucocorticoid signalling  (Bethyl)

Journal: Advanced Science

Article Title: UBX‐390: A Novel Androgen Receptor Degrader for Therapeutic Intervention in Prostate Cancer

doi: 10.1002/advs.202400398

Figure Lengend Snippet: UBX‐390 degrades AR via proteasomal action in a prostate cancer cell line. A) Chemical structures of UBX‐390 and ARV‐110. Immunoblotting results show the AR degradation effect of B) UBX‐390 and ARV‐110 after 20 h of treatment at different doses; and C) 0.1 µ m UBX‐390 and ARV‐110 after treatment for different periods. D) Immunoblotting results showing the UPS in VCaP cells. VCaP cells were pre‐treated with 1 µм bortezomib, MLN4924, or bafilomycin, or 10 µ m pomalidomide for 1 h and then treated with 0.1 µм UBX‐390 for 4 h. E) The AR degradation effect of 0.1 µ m AR degraders, ARV‐110, UBX‐390, and ARCC‐4, and an inhibitor, Enz, in control and CRBN‐knockout CRISPR pool VCaP cell lines. The values for the remaining AR were normalized to those of GAPDH, which served as a loading control. Immunoblotting experiments were conducted independently two times. Data are presented as mean values ± standard error (SE) of the mean. Statistical analyses were performed by Student's t ‐test using GraphPad PRISM 5. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and n.s. means not significant. Enz, enzalutamide; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; AR, androgen receptor.

Article Snippet: Primary antibodies against the following proteins were used for immunoblotting: GR (A303‐490A) and ELL2 (A302505A) from Bethyl Laboratories (Montgomery, TX, USA); PR (sc‐810), VHL (sc‐17780), PCLAF (sc‐390515), and hemagglutinin‐tag (sc‐7392) from Santa Cruz Biotechnology (Dallas, TX, USA); ER (8644), AR (5153S), glyceraldehyde‐3‐phosphate dehydrogenase (2118S), c‐myc (5605S), NKX3.1 (83700S), ERG (97249S), lamin B (17416S), α‐tubulin (2125s), and vinculin (4650S) from Cell Signaling Technology (Danvers, MA, USA); CRBN (NBP1‐91810) from Novus Biologicals (Centennial, CO, USA); CRISP‐3 (AF2397) from R&D Systems (McKinley Place NE, MN, USA); KLK2 (ab152136), SCD1 (ab39969), and BNIP3 (ab109362) from Abcam (Cambridge, UK).

Techniques: Western Blot, Control, Knock-Out, CRISPR

Journal: Advanced Science

Article Title: UBX‐390: A Novel Androgen Receptor Degrader for Therapeutic Intervention in Prostate Cancer

doi: 10.1002/advs.202400398

Figure Lengend Snippet: UBX‐390 leads to potent and stable inhibition of the AR pathway. A) Immunoblotting showing the nuclear receptor selectivity of UBX‐390 and other AR degraders in T47D cells at 24 h after treatment with 0.05 µ m UBX‐390, ARV‐110, ARCC‐4, or Enz. B) Quantitative proteomic analysis was performed to evaluate proteomic changes in VCaP cells. The cells were treated with 0.1 µ m UBX‐390 or DMSO for 4 or 24 h. The lysates obtained were treated with the TMT10plex kit, followed by liquid chromatography‐tandem mass spectrometry‐based proteomic analysis. The volcano plot indicates the protein ranking per abundance ratio (log 2 fold change) for DMSO and UBX‐390, along with the corresponding statistical p ‐value. The non‐axial vertical line represents a fold change of ±1.5, whereas the non‐axial horizontal line indicates the threshold of a significant p ‐value of 0.05. This experiment was performed in triplicate. A total of 7330 proteins were identified in this study. C) Immunoblotting showing the effect of 0.1 µм UBX‐390 or ARV‐110 on AR signaling‐ and prostate cancer‐related proteins in AR‐activated VCaP cells treated with 1 n m DHT. D) Unsupervised hierarchical clustering analysis of DHT‐response genes and E) expression of DHT early‐/late‐response genes in VCaP cells treated with 0.1 µ m ARV‐110 or UBX‐390, in combination with 1 n m DHT, for 24 or 72 h. F) Gene Ontology enrichment analysis of DHT early‐response (top, n = 415) and late‐response (bottom, n = 330) genes. Immunoblotting experiments were conducted independently two times. The proteomics and RNA‐seq experiments were conducted in duplicate. Data are presented as mean values ± SD of the mean. The Reads Per Kilobase per Million mapped reads (RPKM) values were subsequently normalized using Z‐scores. Statistical analyses were performed by Student's t ‐test using GraphPad PRISM 5. **** p < 0.0001. DHT, dihydrotestosterone; Enz, enzalutamide; AR, androgen receptor.

Article Snippet: Primary antibodies against the following proteins were used for immunoblotting: GR (A303‐490A) and ELL2 (A302505A) from Bethyl Laboratories (Montgomery, TX, USA); PR (sc‐810), VHL (sc‐17780), PCLAF (sc‐390515), and hemagglutinin‐tag (sc‐7392) from Santa Cruz Biotechnology (Dallas, TX, USA); ER (8644), AR (5153S), glyceraldehyde‐3‐phosphate dehydrogenase (2118S), c‐myc (5605S), NKX3.1 (83700S), ERG (97249S), lamin B (17416S), α‐tubulin (2125s), and vinculin (4650S) from Cell Signaling Technology (Danvers, MA, USA); CRBN (NBP1‐91810) from Novus Biologicals (Centennial, CO, USA); CRISP‐3 (AF2397) from R&D Systems (McKinley Place NE, MN, USA); KLK2 (ab152136), SCD1 (ab39969), and BNIP3 (ab109362) from Abcam (Cambridge, UK).

Techniques: Inhibition, Western Blot, Liquid Chromatography, Mass Spectrometry, Expressing, RNA Sequencing

Journal: Advanced Science

Article Title: UBX‐390: A Novel Androgen Receptor Degrader for Therapeutic Intervention in Prostate Cancer

doi: 10.1002/advs.202400398

Figure Lengend Snippet: UBX‐390 interferes with DHT‐dependent AR recruitment on the chromatin. A) Assessment of the AR degradation effect of 0.1 µ m AR degraders in the cytosol and nucleus after treatment with 1 n m DHT for 24 and 72 h, followed by nuclear fractionation. B) Density plot of AR ChIP‐seq signals in 0.1 µ m AR degrader‐treated VCaP cells at the indicated genomic regions (10 kb around the center of the AR peak, n = 14213). C) Metagene representations based on AR ChIP‐seq of AR‐occupied regions in the indicated cells. D) Box plot of AR ChIP‐seq counts at the AR peak ( n = 14213). E) Principal component analysis based on AR ChIP‐seq signals. F,G) Representative browser tracks of AR ChIP‐seq and RNA‐seq in the indicated cells at the F) TMPRSS2 and G) KLK2/3/4 loci. Immunoblotting experiments were conducted independently two times. AR ChIP‐seq and RNA‐seq experiments were conducted in duplicate. Data are presented as mean values ± standard deviation (SD) of the mean. AR ChIP‐seq counts were subsequently normalized using Z‐scores. DHT, dihydrotestosterone; Enz, enzalutamide; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; AR, androgen receptor.

Article Snippet: Primary antibodies against the following proteins were used for immunoblotting: GR (A303‐490A) and ELL2 (A302505A) from Bethyl Laboratories (Montgomery, TX, USA); PR (sc‐810), VHL (sc‐17780), PCLAF (sc‐390515), and hemagglutinin‐tag (sc‐7392) from Santa Cruz Biotechnology (Dallas, TX, USA); ER (8644), AR (5153S), glyceraldehyde‐3‐phosphate dehydrogenase (2118S), c‐myc (5605S), NKX3.1 (83700S), ERG (97249S), lamin B (17416S), α‐tubulin (2125s), and vinculin (4650S) from Cell Signaling Technology (Danvers, MA, USA); CRBN (NBP1‐91810) from Novus Biologicals (Centennial, CO, USA); CRISP‐3 (AF2397) from R&D Systems (McKinley Place NE, MN, USA); KLK2 (ab152136), SCD1 (ab39969), and BNIP3 (ab109362) from Abcam (Cambridge, UK).

Techniques: Fractionation, ChIP-sequencing, RNA Sequencing, Western Blot, Standard Deviation

Journal: Advanced Science

Article Title: UBX‐390: A Novel Androgen Receptor Degrader for Therapeutic Intervention in Prostate Cancer

doi: 10.1002/advs.202400398

Figure Lengend Snippet: UBX‐390 exhibits therapeutic activity in xenograft models of prostate cancer. A) Evaluation of the inhibitory effects of ARV‐110, UBX‐390, ARCC‐4, and enzalutamide on cell proliferation of VCaP cells, 6 d post‐treatment. This experiment was performed in duplicate. B) Experimental scheme of the in vivo experimental design of the VCaP xenograft model. ( n = 7 for each group) C) Graph showing tumor growth following vehicle, UBX‐390, or enzalutamide administration to the VCaP xenograft mouse models. D) Representative photographs of subcutaneous tumors derived from the VCaP cells. Scale bar = 10 mm. E) Immunoblotting showing the AR degradation effect of UBX‐390 in tumor tissues extracted from the xenograft model. The proliferation experiments were conducted in duplicate. Data for tumor growth are presented as mean values ± SE of the mean. Statistical analyses were performed by one‐way analysis of variance (ANOVA) tests using GraphPad PRISM 5. **** p < 0.0001, and n.s. means not significant.

Article Snippet: Primary antibodies against the following proteins were used for immunoblotting: GR (A303‐490A) and ELL2 (A302505A) from Bethyl Laboratories (Montgomery, TX, USA); PR (sc‐810), VHL (sc‐17780), PCLAF (sc‐390515), and hemagglutinin‐tag (sc‐7392) from Santa Cruz Biotechnology (Dallas, TX, USA); ER (8644), AR (5153S), glyceraldehyde‐3‐phosphate dehydrogenase (2118S), c‐myc (5605S), NKX3.1 (83700S), ERG (97249S), lamin B (17416S), α‐tubulin (2125s), and vinculin (4650S) from Cell Signaling Technology (Danvers, MA, USA); CRBN (NBP1‐91810) from Novus Biologicals (Centennial, CO, USA); CRISP‐3 (AF2397) from R&D Systems (McKinley Place NE, MN, USA); KLK2 (ab152136), SCD1 (ab39969), and BNIP3 (ab109362) from Abcam (Cambridge, UK).

Techniques: Activity Assay, In Vivo, Derivative Assay, Western Blot

Journal: Advanced Science

Article Title: UBX‐390: A Novel Androgen Receptor Degrader for Therapeutic Intervention in Prostate Cancer

doi: 10.1002/advs.202400398

Figure Lengend Snippet: UBX‐390 showed broad activity against therapy‐resistant AR mutations in the ligand‐binding domain. A) Structure of AR and its activating point mutations. Immunoblotting showing the AR degradation effect of AR degraders, ARV‐110, UBX‐390, and ARCC‐4, and an AR antagonist, enzalutamide, in HEK293 cells with transient expression of B) WT AR or C) various AR mutants, after 24 h of treatment. WT, wild‐type; Enz, enzalutamide; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; AR, androgen receptor.

Article Snippet: Primary antibodies against the following proteins were used for immunoblotting: GR (A303‐490A) and ELL2 (A302505A) from Bethyl Laboratories (Montgomery, TX, USA); PR (sc‐810), VHL (sc‐17780), PCLAF (sc‐390515), and hemagglutinin‐tag (sc‐7392) from Santa Cruz Biotechnology (Dallas, TX, USA); ER (8644), AR (5153S), glyceraldehyde‐3‐phosphate dehydrogenase (2118S), c‐myc (5605S), NKX3.1 (83700S), ERG (97249S), lamin B (17416S), α‐tubulin (2125s), and vinculin (4650S) from Cell Signaling Technology (Danvers, MA, USA); CRBN (NBP1‐91810) from Novus Biologicals (Centennial, CO, USA); CRISP‐3 (AF2397) from R&D Systems (McKinley Place NE, MN, USA); KLK2 (ab152136), SCD1 (ab39969), and BNIP3 (ab109362) from Abcam (Cambridge, UK).

Techniques: Activity Assay, Ligand Binding Assay, Western Blot, Expressing