Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer
- Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, China
- Department of Urology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, China
- Department of Medical Research Center, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, China
- Key Laboratory of the Ministry of Education for Advanced Catalysis Materials, Department of Chemistry, Zhejiang Normal University, China
Figures
Figure 1 with 4 supplements
Z15 specifically inhibits the transcription activity of AR and AR mutants.
(A) Chemical structure of Z15. (B) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in PC-3 cells co-transfected with Renilla, AR, and PSA promoter expression vector plasmids, stimulated by 5 nM DHT, and treated with different concentrations of Z15 for 24 hr. (C) LNCaP, (D) VCaP, (E) and 22Rv1 cells co-transfected with Renilla and PSA promoter expression vector plasmids, stimulated by 5 nM DHT, and treated with different concentrations of Z15 for 24 h. (F) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in LNCaP cells stimulated by 5 nM DHT, and treated with different concentrations of Z15 or ENZa for 24 hr. (G) Dual-luciferase reporter assays to measure MMTV-luc reporter luciferase activities in PC-3 cells co-transfected with Renilla and MMTV promoter expression vector plasmids stimulated by 100 nM Dex, and treated with different concentrations of Z15 for 24 hr. (H) Dual-luciferase reporter assays to measure PSA-luc reporter luciferase activities in PC-3 cells co-transfected with Renilla, AR_T877A mutation, and PSA promoter expression vector plasmids stimulated by 5 nM DHT treated with different concentrations of Z15 for 24 hr. (I) PC-3 cells co-transfected with Renilla, AR_F876L mutation, and PSA promoter expression vector plasmids, treated with different concentrations of Z15 for 24 hr. All experiments were performed in triplicate. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs DHT or Dex group. ENZa, enzalutamide; DHT, dihydrotestosterone; Dex, dexamethasone; Mif, mifepristone.
Figure 1—figure supplement 1
In silico screening procedure.
Figure 1—figure supplement 2
Primary bioactivity evaluation of virtual screened AR inhibitor candidates.
(A) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities stimulated by 5 nM DHT, and treated with 10 μM indicated compounds for 24 hr, in PC-3 cells co-transfected with the Renilla, AR and PSA promoter expression vector plasmids, and (B) Z15 showed repeated strongest AR inhibition activity. (C) Dual-luciferase reporter assay to measure MMTV-luc reporter luciferase activities stimulated by 100 nM Dex, and treated with 10 μM Z15 for 24 hr, in PC-3 cells co-transfected with the Renilla and MMTV promoter expression vector plasmids. Experiments were in triplicate. All results are shown as mean ± s.d. *p<0.05, **p<0.01, ***p<0.001 vs DHT or Dex group. ENZa: Enzalutamide; DHT: dihydrotestosterone; Dex: dexamethasone; Mif: mifepristone.
Figure 1—figure supplement 3
The procedure for the synthesis of compound Z15.
Figure 1—figure supplement 4
Z15 specifically inhibits the transcriptional activity of AR rather than GR, ER and PR.
(A) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in PC-3 cells co-transfected with the Renilla, wt-AR, and PSA promoter expression vector plasmids, stimulated by 5 nM DHT, and treated with different concentrations of Z15 for 24 hr. (B) Dual-luciferase reporter assay to measure MMTV-luc reporter luciferase activities in PC-3 cells (endogenous GR expression cells) co-transfected with the Renilla and MMTV expression vector plasmids, stimulated by 100 nM Dex, and treated with different concentrations of Z15 for 24 hr. (C) Dual-luciferase reporter assay to measure ERE-luc reporter luciferase activities in PC-3 cells co-transfected with the Renilla, ER and ERE expression vector plasmids, stimulated by 100 nM Dien, and treated with different concentrations of Z15 for 24 hr. (D) Dual-luciferase reporter assay to measure GRE-luc reporter luciferase activities in MCF7 cells (endogenous GR expression cells) co-transfected with the Renilla and GRE expression vector plasmids, stimulated by 100 nM Pro, and treated with different concentrations of Z15 for 24 hr. Experiments were in triplicate. All results are shown as mean ± s.d. *p<0.05, **p<0.01, ***p<0.001 vs DHT, or Dex, or Dien, or Pro group. ENZa: Enzalutamide; DHT: dihydrotestosterone; Dex: dexamethasone; Mif: mifepristone; Dien, Dienestrol; Ful, Fulvestrant; Pro, Progesterone.
Figure 2 with 3 supplements
Z15 downregulates AR target genes and ARlevels.
(A) LNCaP cells treated with vehicle, 0.5, or 5 μM Z15 in the presence of 5 nM DHT for 24 hr before performing RNA-sequencing. Heatmap shows the expression levels of AR target genes. (B) The mRNA levels of PSA, PMEPA1, and TMPRSS2 measured by quantitative-PCR and normalized to GAPDH in LNCaP cells treated with vehicle or different concentrations of Z15 in the presence of 5 nM DHT for 24 hr. (C) The mRNA levels of PSA measured by quantitative-PCR and normalized to GAPDH in 22Rv1 and VCaP cells treated with vehicle or different concentrations of Z15 in the presence of 5 nM DHT for 24 hr. (D) Western blot analysis of LNCaP cells treated with indicated concentrations of Z15 in the presence of 5 nM DHT for 24 hr, before cell lysing and determining PSA and AR protein levels. (E) Western blot analysis performed in 22Rv1 cells. (F) Western blot analysis performed in VCaP cells. (G) Western blot analysis of LNCaP cells treated with indicated concentrations of Z15 in the absence of DHT for 24 hr, before cell lysing, and determining AR protein levels. (H) Western blot analysis of 22Rv1 cells treated with indicated concentrations of Z15 in the absence of DHT for 24 hr, before cell lysing, and determining AR protein levels. Experiments were performed in triplicate. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs DHT group.
Figure 2—figure supplement 1
Z15 down regulates AR and ARV7 protein levels.
(A) AR protein quantitative analysis of Figure 2D. (B) PSA protein quantitative analysis of Figure 2D. (C) AR protein quantitative analysis of Figure 2E. (D) ARV7 protein quantitative analysis of Figure 2E. (E) PSA protein quantitative analysis of Figure 2E. (F) AR protein quantitative analysis of Figure 2F. (G) PSA protein quantitative analysis of Figure 2F. (H) Cells were treated with indicated concentration of Z15 in the absence of DHT for 24 hr, then cells were lysed, AR protein levels were measured by western blot analysis, performed in LNCaP cells and (I) AR and ARV7 protein levels were measured by Western blot analysis, performed in 22Rv1 cells. ImageJ was used for protein quantitative analysis.
Figure 2—figure supplement 2
The influence of Z15 and ARV-110 on LNCaP cells global proteomics.
(A) Volcano diagram of differential proteins between 5 μM ARV-110 plus 5 nM DHT treated group and 5 nM DHT treated group. (B) Volcano diagram of differential proteins between 5 μM Z15 plus 5 nM DHT treated group and 5 nM DHT treated group. (C) KEGG analysis of differential proteins between 5 μM ARV-110 plus 5 nM DHT treated group and 5 nM DHT treated group. (D) KEGG analysis of differential proteins between 5 μM Z15 plus 5 nM DHT treated group and 5 nM DHT treated group. Experiments were in triplicates.
Figure 2—figure supplement 3
Z15 have no influence on GR, HSP90, and CDK7 protein levels.
Western blot analysis of 22Rv1 cells treated with indicated concentrations of Z15 for 24 hr, before cell lysing and determining AR, GR, HSP90 and CDK7 protein levels.
Figure 3
Z15 inhibits AR nuclear localization.
(A) Nuclear localization of AR in LNCaP cells treated with vehicle or 5 μM compounds in the presence of 5 nM DHT for 4 h. (B) Quantitative analysis of AR nuclear localization.Experiments were performed in triplicate.
Figure 4 with 3 supplements
Z15 directly binds to AR.
(A) Competitive binding assay to detect binding affinity of R1881, ENZa, and Z15 to AR LBD, 1 nM radioligand [3H] DHT and LNCaP cytosol were used. (B) Biolayer interferometry measurements of Z15 binding to AR LBD. (C) Sensorgram and steady state fitted results of surface plasmon resonance assay to detect binding affinity between Z15 and AF1. Experiments were performed in triplicate.
Figure 4—figure supplement 1
Competitive binding of Z15 to AR-LBD evaluated by AR fluorescence polarization (FP) assay.
PolarScreen Androgen Receptor Competitor Assay kit, Green (Invitrogen, A15880) was used according to manufacturer`s procedure to determine relative in vitro binding affinity of Z15 for the rat AR ligand-binding domain (LBD). Experiments were in triplicates. All results are shown as mean ± s.d. *p<0.05, **p<0.01, ***p<0.001 vs control group.
Figure 4—figure supplement 2
Z15 could bind to AR LBD and AR AF1 directly.
(A) Biolayer interferometry measurements of ENZa binding to AR LBD. (B) Biolayer interferometry measurements of Z15 binding to AR AF1. (C) Biolayer interferometry measurements of UT-34 binding to AR AF1. (D) Biolayer interferometry measurements of ENZa binding to AR LBD. Experiments were performed in triplicate.
Figure 4—figure supplement 3
The influences of ARV-110 and UT-34 on 22Rv1 AR and ARV7 protein levels.
(A) Western blot analysis of 22Rv1 cells treated with indicated concentrations of ARV-110 for 24 hr, before cell lysing and determining AR protein levels. (B) AR protein quantitative analysis of Figure 4—figure supplement 2A. (C) ARV7 protein quantitative analysis of Figure 4—figure supplement 2A. (D) Western blot analysis of 22Rv1 cells treated with indicated concentrations of UT-34 for 24 hr, before cell lysing and determining AR protein levels. (E) AR protein quantitative analysis of Figure 4—figure supplement 2D. (F) ARV7 protein quantitative analysis of Fig Figure 4—figure supplement 2D.
Figure 5 with 1 supplement
Z15 promotes AR degradation in proteasome pathway-dependent manner.
A-C Western blot analysis of AR protein levels, and quantitative-PCR normalized to GAPDH of AR mRNA levels in LNCaP (A), 22Rv1 (B), and VCaP (C) cells treated with indicated concentrations of Z15 in the absence of DHT for 24 hr. (D) Western blot analysis of AR in LNCaP cells treated with 100 μg/mL CHX in the presence or absence of 5 μM Z15 for indicated time points. (E) Western blot analysis of AR protein levels in LNCaP and VCaP cells treated with 5 μM Z15 or/and 5 μM MG 132 for 8 hr. (F) Immunoprecipitation done using anti-AR and immunoblotting with anti-Myc antibody in 22Rv1 cells co-transfected with Myc-tag CW7-UB plasmids treated with or without 5 μM Z15 in the presence of 5 μM Mg132 for 12 hr. Input: immunoblot of lysates probed with AR antibody. Experiments were performed in triplicate. All results are shown as mean ± sd. CHX, cycloheximide.
Figure 5—figure supplement 1
Z15 promotes AR degradation in proteasome pathway-dependent manner.
(A) AR protein quantitative analysis of Figure 5A. (B) AR protein quantitative analysis of Figure 5B. (C) ARV7 protein quantitative analysis of Figure 5B. (D) AR protein quantitative analysis of Figure 5C. (E) AR protein quantitative analysis of Figure 5D. (F) AR protein (LNCaP) quantitative analysis of Figure 5E. (G) AR protein (VCaP) quantitative analysis of Figure 5E.
Figure 6 with 1 supplement
Z15 selectively inhibits proliferation and induces apoptosis of AR-positive CRPC cells.
(A) VCaP, 22Rv1, DU145 and PC-3 cells treated with different concentrations of Z15 or ARV-110 for 72 hr, cell proliferation detected by MTT assay. (B) Crystal violet staining of PC-3 and 22Rv1 cells treated with or without 1 μM Z15 or ARV-110 for 12–14 days, colony numbers were quantified. (C) Patient-derived PCa organoid treated with 1 μM Z15 or DMSO for 7 days, then observed by microscope. (D) Western blot analysis of cleaved PARP protein levels in VCaP cells treated with indicated concentrations of Z15 for 24 hr. (E) Western blot analysis of cleaved PARP protein levels in 22Rv1 cells treated with indicated concentrations of Z15 for 24 hr. Experiments were performed in triplicate. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs DMSO group.
Figure 6—figure supplement 1
Z15 promotes the apoptosis of AR-positive CRPC cell lines.
(A) Western blot analysis of VCaP cells treated with 5 μM Z15 for indicated hours, before cell lysing and determining Cleaved PARP protein levels. (B) Western blot analysis of 22Rv1 cells treated with 5 μM Z15 for indicated hours, before cell lysing and determining Cleaved PARP protein levels. (C) Cleaved PARP protein quantitative analysis of Figure 6D. (D) Cleaved PARP protein quantitative analysis of Figure 6E. (E) Western blot analysis of DU145 cells treated with indicated concentrations of Z15 for 24 hr, before cell lysing and determining Cleaved PARP protein levels.
Figure 7 with 1 supplement
Z15 suppresses 22Rv1 xenografts progress in vivo.
(A) Xenografts arising from 22Rv1 cells treated with blank control, 10, or 20 mg/kg Z15 once a day for 21 days. Mice weighed by electronic scale. (B) Tumor growth monitored every other day. (C) Tumor weight monitored on the last day. (D) Western blot analysis of AR, ARV7, and PSA protein levels in lysed tumor tissues. (E) Immunohistochemical analysis of proliferation (Ki67) and PSA levels in harvested tumors. Scale bar represents 50 μm. Results are shown as mean ± sd. *p<0.05, **p<0.01, ***p<0.001 vs control group.
Figure 7—figure supplement 1
Z15 reduced AR, ARV7 and PSA levels in vivo.
Figure 8 with 3 supplements
Z15 analogs show comparable AR inhibition activity.
(A) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities in LNCaP cells stimulated by 5 nM DHT, and treated with different concentrations of indicated compounds for 24 hr. (B) AR transcription inhibition IC50. (C) Western blot analysis of PSA and AR protein levels of lysed LNCaP cells treated with indicated concentrations of Z15 and its analogs in the presence of 5 nM DHT for 24 hr. Results are shown as mean ± sd. Experiments were performed in triplicate.
Figure 8—figure supplement 1
The chemical structure of seven Z15 analogues.
Figure 8—figure supplement 2
Z15 analogues could inhibit AR transcription activity and reduce AR protein levels.
(A) Dual-luciferase reporter assay to measure PSA-luc reporter luciferase activities stimulated by 5 nM DHT, and treated with 1 μM indicated compounds for 24 hr, in PC-3 cells co-transfected with the Renilla, AR and PSA promoter expression vector plasmids. (B) LNCaP Cells were treated with 5 μM indicated compounds in the presence of 5 nM DHT for 24 hr, then cells were lysed, PSA and AR protein levels were measured by Western blot analysis. Experiments were in triplicate. All results are shown as mean ± s.d. *p<0.05, **p<0.01, ***p<0.001 vs DHT.
Figure 8—figure supplement 3
Z15 analogs downregulate AR and PSA protein levels.
Figure 9
The mechanism of Z15 inhibits the AR pathway and overcomes antiandrogen resistance.
Z15 binds to both AR LBD and AR AF1, decreases AR nuclear translocation, antagonizes AR function, promotes AR and ARVs degradation through the proteasome pathway, so as to overcome AR mutation, AR overexpression, and ARVs-induced antiandrogen resistance.
Additional files
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Supplementary file 1
Supplementary tables described in this article.
(a) The chemical structures of 80 candidate compounds. (b) Primer sequences for qRT-PCR. (c) The spectroscopic data of the Z15 derivatives. (d) Decreased protein profiles of LNCaP cells treated with ARV-110 plus DHT compared to DHT detected by 4D-lael free proteomics. (e) Decreased protein profiles of LNCaP cells treated with Z15 plus DHT compared to DHT detected by 4D-lael free proteomics
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Source data 1
Original gel and blot.
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Source data 2
Figure supplement source data.
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Source data 3
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Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer
eLife 12:e70700.
https://doi.org/10.7554/eLife.70700
