Author response: Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer

Abstract

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics Abstract In patients with castration-resistant prostate cancer (CRPC), clinical resistances such as androgen receptor (AR) mutation, AR overexpression, and AR splice variants (ARVs) limit the effectiveness of second-generation antiandrogens (SGAs). Several strategies have been implemented to develop novel antiandrogens to circumvent the occurring resistance. Here, we found and identified a bifunctional small molecule Z15, which is both an effective AR antagonist and a selective AR degrader. Z15 could directly interact with the ligand-binding domain (LBD) and activation function-1 region of AR, and promote AR degradation through the proteasome pathway. In vitro and in vivo studies showed that Z15 efficiently suppressed AR, AR mutants and ARVs transcription activity, downregulated mRNA and protein levels of AR downstream target genes, thereby overcoming AR LBD mutations, AR amplification, and ARVs-induced SGAs resistance in CRPC. In conclusion, our data illustrate the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment. Editor's evaluation The present study reports the discovery and preclinical evaluation of a novel therapeutic agent for the treatment of castration-resistance prostate cancer through inducing degradation of androgen receptor. The major strength of this study is the identification of a novel lead compound and its interesting in vitro and in vivo activities in prostate cancer models. https://doi.org/10.7554/eLife.70700.sa0 Decision letter eLife's review process Introduction Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer-related death for men in western countries (Siegel et al., 2022; Sung et al., 2021). Advanced PCa initially responds to androgen deprivation therapy (ADT), but invariably fails and recurs as lethal castration-resistant prostate cancer (CRPC) (Harris et al., 2009; Desai et al., 2021). Androgen receptor (AR) signaling plays a crucial role in the progress and survival of CRPC (Dai et al., 2017). Second-generation antiandrogens (SGAs), such as enzalutamide (ENZa), abiraterone, apalutamide, and darolutamide, improve the overall survival time and decline prostate-specific antigen (PSA) levels in patients with CRPC (Sternberg et al., 2020; Armstrong et al., 2019; Smith et al., 2021; Smith et al., 2022; Ryan et al., 2015; de Bono et al., 2011). Despite the initial benefit of these agents, their success in treating CRPC has been eliminated by the emergence of drug resistance. Multiple possible mechanisms for the development of drug resistance have thus far been identified, including mutations in the AR LBD, amplification of AR, expression of AR splice variants (ARVs), and intra-tumoral de novo androgen synthesis (Buttigliero et al., 2015; Robinson et al., 2015; Karantanos et al., 2015). Therefore, more effective therapies are urgently required to conquer the SGAs drug resistance. Several strategies have been implemented to develop novel antiandrogens to circumvent the occurring resistance. The first strategy is to develop new competitive antiandrogens targeting the AR hormone-binding pocket (HBP) site, such as darolutamide (Smith et al., 2022). Another strategy is to target the AR signaling axis beyond the HBP site, which includes activation function-1 (AF1), activation function-2, binding function 3, and the DNA binding site through active compounds, such as EPI-001, VPC-14449 (Caboni and Lloyd, 2013). Recently, down-regulating both AR protein and AR mRNA levels has attracted attention due to their potential in the discovery and development of new antiandrogens. The most exciting progress is AR degradation based on the proteolysis targeting chimeras (PROTACs) concept, with various of these AR PROTACs developed with a DC50 (drug concentration that results in 50% protein degradation) potency up to 1 nM. However, low cell permeability, poor pharmacokinetic properties, and complex chemical structures may restrict the clinical application of PROTAC drugs (He et al., 2020). What’s more, LBD-targeted AR PROTACs cannot degrade ARVs which were associated with unfavorable clinical outcomes in patients with CRPC (Fettke et al., 2020). The selective estrogen receptor degrader fulvestrant approved by the FDA in 2002 expanded treatment choices for advanced breast cancer (Bross et al., 2003), which gave rise to next-generation novel degraders with promising antitumor activity in recent years (Nardone et al., 2019). Bradbury et al., 2011 suggested that similar specific downregulation or degradation of AR might be proved beneficial in the treatment of CRPC. Therefore, selective AR degraders (SARDs) which could synthetically degrade and antagonize AR may be an efficient strategy to overcome the drug resistance in the antiandrogen therapy of CRPC. Based on structural modification of the AR antagonists and the tissue-selective AR agonist enobosarm, Miller et al. designed a series of SARDs, namely UT-155, UT-69, and UT-34, which could induce AR ubiquitin-proteasome degradation via binding to AF-1 of the AR to reduce its stability (Ponnusamy et al., 2017; Ponnusamy et al., 2019; Hwang et al., 2019). Notably, the degradation potency of these compounds for ARVs is quite limited. In the present study, we determined that Z15 screened by rational drug design as an AR antagonist and degrader via direct binding to the AR LBD and AR AF1, could overcome AR LBD mutations, AR amplification, and ARVs-induced SGAs resistance of CRPC in vitro and in vivo. Results Identifying Z15 as an AR inhibitor To develop novel AR inhibitors and overcome antiandrogen resistance, we previously constructed a common molecular characteristic pharmacophore model, and screened ~7.5 million compounds from the ZINC lead-like database and ChemDiv database. About 47,202 compounds matched more than four features of the filtering model. Next, these compounds were docked into the HBP of the antagonistic AR. Then, compounds with the top 1000 docking scores were chosen for ADMET prediction by Discovery Studio v3.5. Finally, 80 hits with high drug-likeness were selected and purchased for further bioactivity evaluation (Figure 1—figure supplement 1 and Supplementary file 1a). To preliminarily evaluate the influences for AR transcriptional activity of these 80 candidates, human prostate cancer cells PC-3 co-transfected with wild-type AR (wt-AR) and PSA-luc were incubated with 5α-dihydrotestosterone (DHT) and 10 μM candidate compounds for 24 h. The cell lysates were collected and AR transcriptional activity was detected by dual-luciferase reporter assay. We identified 19 compounds that showed more than 25% AR transcription inhibition activity, among which compound Z15 (structure shown in Figure 1A) exhibited the most potent AR inhibition activity (Figure 1—figure supplement 2A). Nevertheless, the glucocorticoid receptor (GR) transcription inhibition activity of Z15 was quite feeble (Figure 1—figure supplement 2B–C). Figure 1 with 4 supplements see all Download asset Open asset 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. Z15 selectively suppresses AR and AR mutant transcriptional activity To further investigate the AR inhibition potency of Z15, we optimized the synthesis route and prepared a sufficient amount of Z15 (Figure 1—figure supplement 3). Next, we performed a dual-luciferase reporter assay in several human PCa cell lines including wt-AR-transfected PC-3 and LNCaP cells. The results indicated that Z15 could inhibit DHT-induced transcriptional activities of both exogenous and endogenous AR in a dose-dependent manner (Figure 1B–C). Unexpectedly, Z15 showed potent AR transcription inhibition activity in AR overexpression and ENZa-insensitive VCaP cells (Figure 1D). In another ENZa resistance 22Rv1 cells which naturally express AR and ARV7, Z15 also inhibited DHT-activated AR transcriptional activity (Figure 1E). Moreover, the AR transcription inhibition IC50 (half-maximal inhibitory concentration) of Z15 in LNCaP cells was ~0.22 μM, which was comparable to ENZa (Figure 1F). We further detected the selectivity of Z15 in GR-positive PC-3 cells, the results indicated that Z15 hardly inhibited dexamethasone activated GR transcriptional activity compared to the GR antagonist mifepristone (Figure 1G). Then, we compared AR, GR, estrogen receptor (ER), and progesterone receptor (PR) transcription inhibition activities of Z15 by dual-luciferase reporter assay. The transcription inhibition IC50 of Z15 was 0.41 μM for AR (Figure 1—figure supplement 4A), over 20 μM for GR and ER (Figure 1—figure supplement 4B–C), and 9.29 μM for PR (Figure 1—figure supplement 4D), which suggests that Z15 is a highly selective AR inhibitor. AR LBD point mutations such as AR T877A (a flutamide-resistant mutation) and AR F876L (ENZa- and apalutamide-resistant mutation), are key causes leading to antiandrogen resistance. Dual-luciferase reporter assay results indicated Z15 could efficiently inhibit DHT-induced both AR T877A and AR F876L transcriptional activities (Figure 1H–I). Taken together, these data illustrate Z15 as a potent selective AR inhibitor both for wild-type and mutated ARs. Z15 inhibits the AR pathway Next, we assessed the influence of Z15 on LNCaP cells transcriptome by RNA-sequencing analysis. Obviously, Z15 dose-dependently inhibited a series of DHT-activated AR downstream genes (Figure 2A). Then, we detected three canonical AR downstream-regulated genes (PSA, PMEPA1, and TMPRSS2) by quantitative real-time PCR (qRT-PCR) assay. The results revealed that Z15 significantly inhibited the mRNA expression levels of these genes (Figure 2B), consistent with the findings of RNA-sequencing. Furthermore, Z15 also decreased DHT-induced PSA mRNA levels in the antiandrogen resistance 22Rv1 and VCaP cells (Figure 2C). Figure 2 with 3 supplements see all Download asset Open asset 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. We further detected the influence of Z15 on AR and PSA protein levels in LNCaP cells. As demonstrated in Figure 2D, Z15 reduced DHT-activated PSA protein levels significantly, which was in line with the qRT-PCR analysis. Surprisingly, AR protein levels were also downregulated by Z15, quite different from the effects of ENZa (Figure 2—figure supplement 1A–B). Notably, Z15 potently inhibited PSA and AR protein levels in ENZa resistance 22Rv1 and VCaP cells (Figure 2E–F and Figure 2—figure supplement 1C–G). Then, we evaluated the AR DC50 of Z15 in LNCaP and 22Rv1 cells. The AR DC50 of Z15 in LNCaP cells was 1.05 μM (Figure 2G and Figure 2—figure supplement 1H), while in 22Rv1 cells it was 1.16 μM and the ARV7 DC50 was 2.24 μM (Figure 2H and Figure 2—figure supplement 1I). In addition, we performed a 4D-label free proteomics study to analyze the effect of Z15 on global protein levels in LNCaP cells. Among 5334 quantifiable proteins, AR LBD-targeted PROTAC molecule ARV-110 significantly reduced 34 proteins and Z15 downregulated 69 proteins compared to the DHT group (Figure 2—figure supplement 2 file 1d-e). Both Z15 and ARV-110 reduced AR, KLK3, and TMPRSS2 protein levels significantly (Figure 2—figure supplement 2A–B). KEGG analysis also proved that these two compounds had a similar influence on the functional pathways (Figure 2—figure supplement 2C–D). Additionally, to verify the specificity of Z15 downregulated AR protein levels, we chose 3 AR pathway related but independent proteins GR, HSP90 (AR chaperonin), and cyclin-dependent kinases 7 (CDK7) as controls. Western blot analysis indicated that Z15 has no influence on GR, HSP90, and CDK7 protein levels in 22Rv1 cells (Figure 2—figure supplement 3). Collectively, these data suggest that Z15 is a novel specific AR pathway inhibitor, which may play a role as an AR antagonist as well as an AR and ARV7 degrader. Z15 inhibits DHT-induced AR nuclear translocation Androgen-binding initiates AR activation, induces its conformational change, and reveals the nuclear localization signal of AR. The hormone-bound AR dimerizes and translocates to the nucleus, where it binds to DNA and interacts with a series of transcriptional coregulators to regulate target gene expression. Accordingly, we investigated whether Z15 disturbed androgen-induced AR nuclear translocation. As shown in Figure 3A–B, the DHT treatment could promote the importing of AR into the nuclear compared to untreated group, while both ENZa and Z15 blocked DHT-induced AR nuclear translocation. This result proves that Z15 can inhibit DHT-induced AR nuclear translocation. Figure 3 Download asset Open asset 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. Z15 binds directly to AR LBD and AR AF1 Since the chemical structure of Z15 is remarkably different from that of previously reported AR antagonists, we next evaluated whether Z15 directly binds to AR in a similar manner as ENZa. The AR competitive binding assay was performed to demonstrate the direct interaction between Z15 and AR, whereby compounds in competition with the radioligand [3H] DHT in cytosolic lysates from LNCaP cells were measured. Synthetic androgen R1881 displayed strong binding potency to AR with an IC50 value of 0.45 nM, which indicated the feasibility of this assay system. The binding affinity between ENZa and AR was 121.2 nM. Interestingly, Z15 showed a comparable binding affinity to ENZa, with an IC50 value of 63.3 nM (Figure 4A). In addition, our fluorescence polarization assay demonstrated Z15 could compete with androgen binding to AR LBD (Figure 4—figure supplement 1). Besides, the biolayer interferometry (BLI) measurement also revealed that both ENZa and Z15 possess AR LBD binding ability (Figure 4B, Figure 4—figure supplement 2A). These data suggested that Z15 could antagonize AR by directly targeting the LBD region. AR LBD targeted compound ARV-110 has been shown as an efficient AR degrader in preclinical research, however, it could not induce ARV7 degradation in 22Rv1 cells (Figure 4—figure supplement 3A–C). Since Z15 could degrade both AR and ARV7, we wondered if Z15 could also bind to other regions of AR to induce ARV7 degradation. Hence, we investigated the binding affinity between Z15 and AR AF1, as AF1 is an important drug target region of AR. The surface plasmon resonance assay indicated that Z15 could directly bind to AR AF1 with a KD value of 0.93 μM (Figure 4C). Z15 was also detected to potently bind to AR AF1 with a comparable binding affinity to AR AF1 inhibitor UT-34 by BLI assay (Figure 4—figure supplement 2B–C). Unexpectedly, UT-34 could not induce ARV7 degradation in 22Rv1 cells from western blot analysis (Figure 4—figure supplement 3D–F). As a control, we did not find any binding potency between AR AF1 and ENZa even at 200 μM (Figure 4—figure supplement 2D). These data illustrate that Z15 potently inhibits ARV7 by directly binding to AR AF1. Figure 4 with 3 supplements see all Download asset Open asset 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. Z15 promotes AR degradation through the proteasome pathway We have shown that Z15 could reduce AR and ARV7 protein levels and conjectured that it is an AR degrader. To confirm this hypothesis, we detected the influence of Z15 on AR protein and mRNA levels in LNCaP cells without DHT treatment. Certainly, Z15 reduced AR protein levels in a dose-dependent manner without influencing the AR mRNA levels (Figure 5A and Figure 5—figure supplement 1A). Moreover, we observed similar effects of Z15 on AR protein and mRNA levels in ENZa resistance cell lines 22Rv1 (Figure 5B and Figure 5—figure supplement 1B–C) and VCaP cells (Figure 5C and Figure 5—figure supplement 1D). Western blot analysis for AR in LNCaP cells treated with protein synthesis inhibitor cycloheximide, showed that Z15 accelerated AR degradation (Figure 5D and Figure 5—figure supplement 1E). These data indicate that Z15 is indeed an AR degrader. Figure 5 with 1 supplement see all Download asset Open asset 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. The ubiquitin-proteasome pathway (UPP) is the main participant that regulates intracellular protein degradation. To explore whether Z15 promoted AR degradation through UPP, LNCaP cells were treated with Z15 in the presence or absence of proteasome inhibitor MG132. Indeed, Z15 reduced the AR protein levels after 8 hr treatment, while AR protein levels reduction was counteracted by MG132. Similarly, Z15 induced AR protein decline was also counteracted by MG132 in VCaP cells (Figure 5E and Figure 5—figure supplement 1F–G). Furthermore, Z15 treatment strikingly induced ubiquitination of AR (Figure 5F). Together, these results indicate that Z15 degrades AR through the UPP. Z15 inhibits proliferation and induces apoptosis in AR-positive CRPC cell lines As Z15 exhibited clear AR and ARV7 inhibition and degradation potency, we next investigated the effects of Z15 on cell proliferation activity in AR-positive CRPC cell lines VCaP and 22Rv1 cells. In VCaP cells, Z15 (IC50=1.37 μM) showed comparable proliferation inhibition potency with positive control ARV-110 (IC50=0.86 μM). However, in ARV7-positive 22Rv1 cells, the proliferation inhibition activity of Z15 (IC50=3.63 μM) was much stronger than that of ARV-110 (IC50=14.85 μM). Both Z15 and ARV-110 displayed weak inhibition effects on the proliferation activity of AR-negative PC-3 and DU145 cells (Figure 6A). To estimate the effects of Z15 on CRPC cell clonogenic activity, we exposed 22Rv1 and PC-3 cells to 1 μM Z15 or ARV-110 for 2 weeks. As a result, Z15 significantly decreased the 22Rv1 cell colony numbers compared to both negative control and ARV-110, whereas both Z15 and ARV-110 showed no influence on the PC-3 cell colony numbers (Figure 6B). Thus, we proved that through blocking and degrading AR, Z15 could selectively inhibit the proliferation of AR and ARV7 positive CRPC cell lines. Furthermore, based on PCa patient tumor derived tissues, we cultured PCa organoids and treated the organoids with 1 μM Z15 for 7 days. The results indicated that Z15 significantly inhibited PCa organoids proliferation compared to the control group (Figure 6C). What’s more, western blot analysis indicated that Z15 also promoted the apoptosis of VCaP and 22Rv1 cells in a dose-dependent manner and time-dependent manner, while Z15 showed no influence on the apoptosis of AR negative DU145 cells (Figure 6D–E and Figure 6—figure supplement 1A–E). Figure 6 with 1 supplement see all Download asset Open asset 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. Z15 inhibits CRPC xenografts growth Our previous experiments proved that Z15 is a selective AR degrader and antagonist with excellent anti-PCa activity in vitro. To evaluate the PCa inhibition activity of Z15 in vivo, we established subcutaneous xenograft tumor models of 22Rv1 cells in the flanks of male BALB/c nude mice. When tumor volumes reached an average size of 50~100 mm3, animals were treated with vehicle control, 10 mg/kg Z15, or 20 mg/kg Z15 for 21 days intragastrically once daily. There were no measurable side effects observed in Z15-treated mice as assessed by the mice body weight (Figure 7A). Treatment of mice with 10 and 20 mg/kg Z15 both suppressed 22Rv1 tumor progression and decreased the tumor weight significantly (Figure 7B–C). In addition, western blot analysis indicated that AR, ARV7, and PSA protein levels in the tumor tissues were significantly declined in both 10 and 20 mg/kg Z15 treatment groups (Figure 7D, Figure 7—figure supplement 1A–C). Immunohistochemistry analysis also revealed that Z15 reduced the Ki-67 and PSA protein levels in tumor tissues (Figure 7E). Taken together, our data indicate that Z15 could inhibit the growth of CRPC both in vitro and in vivo. Figure 7 with 1 supplement see all Download asset Open asset 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. Identifying Z15 analogs as AR inhibitor Since Z15 showed inspiring CRPC inhibition potency, we searched the SciFinder and ZINC database to seek Z15 chemical structure analogs. Seven compounds (Figure 8—figure supplement 1, Supplementary file 1c) with more than 80% similarity to Z15 were eventually obtained for further bioactivity evaluation. Dual-luciferase reporter assay indicated that most of these compounds pronouncedly inhibited DHT-activated AR transcriptional activity at 1 μM except for ZL-1 (Figure 8—figure supplement 2A), suggesting that the dimethyl isoxazole group plays an indispensable role in the AR inhibitory activity of Z15 and its analogs. Western blot analysis revealed that these active molecules also reduced AR and DHT-induced PSA protein levels (Figure 8—figure supplement 2B). Furthermore, we detected the AR transcription inhibition IC50 of six active Z15 analogs. Among these molecules, ZL-2 and ZL-4 exhibited the strongest AR transcription inhibition potency, while others also showed comparable AR inhibition activity compared to Z15 (Figure 8A–B). Western blot analysis revealed that these active molecules could reduce AR and PSA protein levels in a dose-dependent manner. Notably, some of these compounds showed stronger AR downregulation activity than Z15 (Figure 8C and Figure 8—figure supplement 3A–D). Together, these results indicate that through chemical structural modification to Z15, more and more selective AR degraders with stronger AR inhibition activity might be found in the near future. Figure 8 with 3 supplements see all Download asset Open asset 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. Discussion SGAs are becoming more prominent in the clinical treatment of patients with CRPC. However, drug resistance caused by AR mutation, AR amplification, and ARVs, has been widely reported to restrict the clinical benefits of these therapies (Buttigliero et al., 2015; Robinson et al., 2015). AR remains a crucial target for CRPC therapeutic development because of its key function in the progress of CRPC. In this study, we identified a compound Z15 that selectively inhibited AR transcriptional activity and significantly downregulated AR target genes at the mRNA and protein levels. Further studies proved that Z15 could bind directly to both AR LBD and AR AF1, so as to decrease androgen-induced AR nuclear translocation, which confirmed Z15 as an AR antagonist. Moreover, Z15 could also degrade AR and ARV7 through the proteasome pathway (Figure 9). Importantly, several Z15 analogs exhibited stronger AR inhibition and downregulation potency than Z15, suggesting that Z15 is a promising lead compound for further chemical structure optimization. Figure 9 Download asset Open asset 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. AR amplification is a common phenomenon in CRPC patients undergoing antiandrogens treatment. Our data showed that ENZa could hardly downregulate DHT-induced PSA levels in AR-overexpressed VCaP cells, which means that AR antagonizing method is too faint to overcome the drug resistance caused by AR amplification. A rational way to solve this p