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Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by specific genomic regions known as super-enhancers (SEs). The robust transcription of genes at such SEs is enabled by the formation of phase-separated condensates by transcription factors and coactivators with intrinsically disordered regions. The androgen receptor (AR), the main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the transcriptional coactivator mediator complex subunit 1 (MED1) to SEs in androgen-dependent PCa cells, thereby promoting oncogenic transcriptional programs. In this work, we reveal that full-length AR forms foci with liquid-like properties in different PCa models. We demonstrate that foci formation correlates with AR transcriptional activity, as this activity can be modulated by changing cellular foci content chemically or by silencing MED1. AR ability to phase separate was also validated in vitro by using recombinant full-length AR protein. We also demonstrate that AR antagonists, which suppress transcriptional activity by targeting key regions for homotypic or heterotypic interactions of this receptor, hinder foci formation in PCa cells and phase separation in vitro. Our results suggest that enhanced compartmentalization of AR and coactivators may play an important role in the activation of oncogenic transcription programs in androgen-dependent PCa.

The isolation of proteins of interest from cell lysates is an integral step to study protein structure and function. Liquid chromatography is a technique commonly used for protein purification, where the separation is performed by exploiting the differences in physical and chemical characteristics of proteins. The complex nature of proteins requires researchers to carefully choose buffers that maintain stability and activity of the protein while also allowing for appropriate interaction with chromatography columns. To choose the proper buffer, biochemists often search for reports of successful purification in the literature; however, they often encounter roadblocks such as lack of accessibility to journals, non-exhaustive specification of components and unfamiliar naming conventions. To overcome such issues, we present PurificationDB (https://purificationdatabase.herokuapp.com/), an open-access and user-friendly knowledge base that contains 4732 curated and standardized entries of protein purification conditions. Buffer specifications were derived from the literature using named-entity recognition techniques developed using common nomenclature provided by protein biochemists. PurificationDB also incorporates information associated with well-known protein databases: Protein Data Bank and UniProt. PurificationDB facilitates easy access to data on protein purification techniques and contributes to the growing effort of creating open resources that organize experimental conditions and data for improved access and analysis. Database URL https://purificationdatabase.herokuapp.com/

Prostate cancer (PCa) patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer (CRPC). Targeting the androgen receptor (AR) Binding Function-3 (BF3) site offers a promising option to treat CRPC. However, BF3 inhibitors have been limited by poor potency or inadequate metabolic stability. Through extensive medicinal chemistry, molecular modeling, and biochemistry, we identified 2-(5,6,7-trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide (VPC-13789), a potent AR BF3 antagonist with markedly improved pharmacokinetic properties. We demonstrate that VPC-13789 suppresses AR-mediated transcription, chromatin binding, and recruitment of coregulatory proteins. This novel AR antagonist selectively reduces the growth of both androgen-dependent and enzalutamide-resistant PCa cell lines. Having demonstrated in vitro efficacy, we developed an orally bioavailable prodrug that reduced PSA production and tumor volume in animal models of CRPC with no observed toxicity. VPC-13789 is a potent, selective, and orally bioavailable antiandrogen with a distinct mode of action that has a potential as novel CRPC therapeutics.

HDAC inhibition has been shown to induce pharmacological BRCAness in cancer cells with proficient DNA repair activity. This provides a rationale for exploring combination treatments with HDAC and PARP inhibition in cancer types that are insensitive to single-agent PARP inhibitors. Here, we report the concept and characterization of a novel bifunctional PARP inhibitor (kt-3283) with dual activity towards PARP1/2 and HDAC enzymes in Ewing sarcoma cells. Compared to the FDA-approved PARP (olaparib) and HDAC (vorinostat) inhibitors, kt-3283 displayed enhanced cytotoxicity in Ewing sarcoma models. The kt-3283-induced cytotoxicity was associated with a strong S and G2/M cell cycle arrest in the nanomolar concentration range and elevated DNA damage as assessed by γH2AX tracking and comet assays. In three-dimensional spheroid models of Ewing sarcoma, kt-3283 showed efficacy in lower concentrations than olaparib and vorinostat and kt-3283 inhibited colonization of Ewing sarcoma cells in anex vivolung metastasis model. In summary, our data demonstrates the preclinical justification for studying the benefit of dual PARP and HDAC inhibition in the treatment of Ewing sarcoma in a clinical trial and provides proof-of-concept for a bi-functional single-molecule therapeutic strategy.

Background: Hedgehog (Hh) signaling pathway plays a fundamental role in the early stages of development by regulating morphogenesis. GLI transcription factors are drivers of this signaling pathway by regulating the expression of growth related genes. Hyperactivation of GLI proteins have been associated with several cancers including medulloblastoma, glioblastoma, ovarian, prostate and breast cancers. Our study in breast cancer (BCa) suggests transcriptional activation of GLI3 in estrogen receptor alpha (ERα) positive cells upon estradiol stimulation. Loss of ERα greatly decreases GLI3 protein stability and stimulation with estradiol significantly increases GLI3 stability in ER-positive BCa cells. We have discovered that GLI3 forms nuclear complexes with ERα upon estradiol stimulation and that the loss of GLI3 reduces BCa cells growth. Therefore, we hypothesize that ERα-GLI3 complex orchestrates BCa transcriptome required for cell growth and development. Our study focuses on characterizing ERα mediated activation of GLI3 in BCa cells and the possible compensation by other steroid receptors such as the androgen receptor (AR) and the glucocorticoid receptor (GR) in ER-negative BCa cells. Method: We examined ERα- GLI transcriptional activity by RNA sequencing upon stimulation with estradiol and inhibition of GLI activity by GANT61 in ER-positive MCF7 cells. We also identified ERα domains that are essential for GLI3 binding by immunoprecipitation and proximity ligation assay (PLA). To identify ERα-GLI3 interactome, we have optimized Rapid Immunoprecipitation Mass Spectrometry of Endogenous protein (RIME). We also examined the role of GLI3 in ER-negative BCa cell growth and characterized the role of AR and GR in activating GLI3 by luciferase reporter assay.Results: Inhibition of GLI DNA binding by GANT61 reduced the expression of GLI regulated (CDC20, CDK1, UBE2C) and modified expression of ERα regulated (FOXM1, SPC24, KIF20A, BIRC5) genes, suggesting cooperative role of ERα-GLI3 in mediating growth and metastasis. The optimized RIME assay suggests immunoprecipitation of ERα with GLI3 in BCa cells upon estradiol induction. Immunoprecipitation and PLA studies suggest that ERα-N terminal, DNA binding and C-terminal domains interact with GLI3. We also showed that knockdown of GLI3 reduces growth in ER negative BCa cells and that AR or GR stimulation by dihydrotestosterone and dexamethasone respectively, are important for GLI3 transcriptional activity. Conclusion: Collectively, our results suggest a new role of steroid receptors in regulating GLI oncogenic transcriptional activity in BCa, leading to new therapeutic possibilities for patients. Citation Format: Shabnam Massah, Maria Guo, Jane Foo, Ralph Buttyan, Artem Cherkasov, Nada Lallous. Characterization of steroid receptor mediated activation of GLI as driving force of breast cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3080.

Lin28 is a pluripotency factor that regulates cancer cell stem-like phenotypes to promote cancer development and therapy-resistant tumor progression. It acts through its cold shock domain and zinc knuckle domain (ZKD) to interact with the Let-7 pre-microRNA and block Let-7 biosynthesis. Chemical inhibition of Lin28 from interacting with Let-7 presents a therapeutic strategy for cancer therapy. Herein, we present the computer-aided development of small molecules by in silico screening 18 million compounds from the ZINC20 library, followed by the biological validation of 163 predicted compounds to confirm 15 new Lin28 inhibitors. We report three lead compounds, Ln7, Ln15, and Ln115, that target the ZKD of both Lin28A and Lin28B isoforms and block Lin28 from binding Let-7. They restore Let-7 expression and suppress tumor oncogenes such as SOX2 in cancer cells and show strong inhibitory effects on cancer cell stem-like phenotypes. However, minimal impacts of these compounds were observed on Lin28-negative cells, confirming the on-target effects of these compounds. We conclude from this study the discovery of several new Lin28 inhibitors as promising candidate compounds that warrant further drug development into potential anticancer therapies.

Simple Summary DNA topoisomerase II (TOP2) is a drug target for many types of cancers. However, clinically used TOP2 inhibitors not only kill cancer cells, but also damage normal cells, and can even give rise to other types of cancers. To discover new TOP2 inhibitors to more effectively treat cancer patients, we have applied computer-aided drug design technology to develop several TOP2 inhibitors that can strongly inhibit cancer cell growth but exert low side effects. Results of one exemplary compound are presented in this study. It shows several promising drug-like properties that can be potentially developed into anticancer drugs. Abstract Clinically used topoisomerase II (TOP2) inhibitors are poison inhibitors that induce DNA damage to cause cancer cell death. However, they can also destroy benign cells and thereby show serious side effects, including cardiotoxicity and drug-induced secondary malignancy. New TOP2 inhibitors with a different mechanism of action (MOA), such as catalytic TOP2 inhibitors, are needed to more effectively control tumor growth. We have applied computer-aided drug design to develop a new group of small molecule inhibitors that are derivatives of our previously identified lead compound T60. Particularly, the compound T638 has shown improved solubility and microsomal stability. It is a catalytic TOP2 inhibitor that potently suppresses TOP2 activity. T638 has a novel MOA by which it binds TOP2 proteins and blocks TOP2–DNA interaction. T638 strongly inhibits cancer cell growth, but exhibits limited genotoxicity to cells. These results indicate that T638 is a promising drug candidate that warrants further development into clinically used anticancer drugs.

Prostate cancer patients undergoing androgen deprivation therapy almost invariably develop castration-resistant prostate cancer. Resistance can occur when mutations in the androgen receptor (AR) render anti-androgen drugs ineffective or through the expression of constitutively active splice variants lacking the androgen binding domain entirely (e.g., ARV7). In this study, we are reporting the discovery of a novel AR-NTD covalent inhibitor 1-chloro-3-[(5-([(2S)-3-chloro-2-hydroxypropyl]amino)naphthalen-1-yl)amino]propan-2-ol (VPC-220010) targeting the AR-N-ter-minal Domain (AR-NTD). VPC-220010 inhibits AR-mediated transcription of full length and truncated variant ARV7, downregulates AR response genes, and selectively reduces the growth of both full-length AR-and truncated AR-dependent prostate cancer cell lines. We show that VPC-220010 disrupts interactions between AR and known coactivators and coregulatory proteins, such as CHD4, FOXA1, ZMIZ1, and several SWI/SNF complex proteins. Taken together, our data suggest that VPC-220010 is a promising small molecule that can be further optimized into effective AR-NTD inhibitor for the treatment of CRPC., Canadian Institutes of Health Research

Numerous cancers, including prostate cancer (PCa), have been shown to be addicted to transcription programs driven by specific genomic sites known as superenhancers (SEs). Recently, it has been proposed that the robust transcription of genes at such SEs is enabled by the formation of phase-separated condensates by transcription factors and coactivators with intrinsically disordered regions. The androgen receptor (AR), the main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the transcriptional coactivator MED1 to SEs in androgen-dependent prostate cancer cells, thereby promoting oncogenic transcriptional programs. In this work, we show that AR-rich, liquid-like foci form in prostate cancer models upon androgen stimulation. We reveal that foci formation correlates with AR transcriptional activity, which can be modulated by changing cellular foci content genetically or chemically. We also demonstrate that the transcriptional coactivator MED1 plays an essential role in the formation of transcriptionally active AR-rich foci and that AR antagonists that block cofactor recruitment or DNA binding hinder foci formation and thus AR transcriptional activity. The liquid like properties of the condensates were also validated in-vitro by using recombinant AR protein. Using clinical specimens, we detected the interaction between AR and MED1 in advanced forms of prostate cancer and not in benign tissues. These results suggest that enhanced compartmentalization of AR and coactivators at SEs may play an important role in the activation of oncogenic transcription programs in androgen-dependent PCa. A better understanding of the assembly and the regulation of these AR-rich compartments may provide novel therapeutic options for PCa by targeting downstream events of androgen activation and DNA binding of AR. Citation Format: Fan Zhang, Maitree Biswas, Joseph Lee, Shreyas Lingadahalli, Samantha Wong, Christopher Wells, Neetu Saxena, Bei Sun, Ana K. Parra-Nuñez, Christophe Sanchez, Jane Foo, Novia Chan, Lauren Ung, Nabeel Khan, Umut Berkay Altıntaş, Jennifer M. Bui, Yuzhuo Wang, Ladan Fazli, Paul S. Rennie, Nathan Lack, Artem Cherkasov, Martin E. Gleave, Joerg Gsponer, Nada Lallous. Dynamic phase separation of the androgen receptor and its coactivators to regulate gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5729.

Background: MYC oncoproteins are important drivers of human cancers. More specifically, NMYC is amplified and overexpressed in neuroendocrine prostate cancer (NEPC). NMYC elicits its oncogenic effects by forming a heterodimer with MAX. This complex binds to regulating elements and activates the transcription of MYC target genes that play roles in tumor growth and progression. It is well known that members of the MYC family of proteins make compelling targets for cancer treatments. However, to this date, no tangible drug candidates have been developed into the clinics for MYC. In previous studies utilizing a rational computer-aided drug discovery (CADD) approach, we identified VPC-70551 as our most active hit. Subsequent optimization with the VPC-70551 scaffold has led to the identification of a new series of compounds. Using in silico drug screening followed by functional validation, we identified a small molecule inhibitor, VPC-70619, that exhibits higher microsomal stability and is better absorbed and tolerated orally than most MYC inhibitors described in the literature. Method: We used a transcriptional reporter assay to determine the effect of VPC-70619 on MYC-mediated transcription. To validate on-target effect, we evaluated the effect of VPC-70619 treatment on MYC-positive (LASCPC-01, NCIH660, LNCaP N-MYC, 22Rv1 N-MYC) and MYC-negative (HO15.19) cancer cells. We also evaluated the effect of VPC-70619 on MYC/MAX interaction by PLA and on DNA binding by BLI. The direct binding between recombinant MYC/MAX protein complex and VPC-70619 was evaluated by MST. Results: VPC-70619 inhibited MYC transcriptional activity in dose dependent manner and the proliferation of MYC-positive cell lines. VPC-70619 did not interfere with MYC/MAX interaction however it blocked the complex interaction with DNA. We confirmed the direct binding of VPC-70619 to the purified MYC/MAX complex by using MST. Conclusion: This project presents the identification of a new MYC inhibitor that blocks the transcriptional activity of this oncogene and elucidates the molecular mechanism of action of this inhibitor. Our findings help prelude the development and discovery of more effective treatments for NEPC patients. Citation Format: Jane Foo, Anh-Tien Ton, Kriti Singh, Fuqiang Ban, Helene Morin, Joseph Lee, Eric LeBlanc, Nada Lallous, Artem Cherkasov. Structure-based development of a novel MYC inhibitor for neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5731.

Background: The nuclear steroid receptor superfamily encompasses a group of proteins best known fortheir functions as primary transcription factors that are conditionally active when bound to a ligand.Here, we show that a prominent member of this family, the estrogen receptor [ER-α] have a secondaryfunction of activating the Gli family of transcription factors. Gli is recognized as the mediator of activeHedgehog (Hh) signaling and plays an important role in cell development and growth. Gli activity isregulated by a post-translational proteolytic process that is suppressed by Hedgehog signaling.Previously we found that in prostate cancer, the ligand activated androgen receptor [AR] recognizes andbinds to Gli proteins at their Protein Processing Domains. This binding stabilizes Gli proteins in their un-proteolyzed active form and bypasses the Hedgehog signaling, thus promoting cancer progressionthrough non-canonical activation of Gli proteins. Due to the high similarity between AR and ER, wehypothesized that a similar Gli regulation could play a role in Breast Cancer (BrCA) progression. We thustested the ability of human ER-α to bind and activate Gli in BrCa and evaluated the role of this pathwayin tumor cell growth. Methods: we measured Gli activity in 293T and BrCa cells (MCF7, T47D, MDA-MB-453) in presence andabsence of steroid ligand using Gli-luciferase reporter assay. We evaluated the interaction between Gli3and ER-α by co-immunoprecipitation and proximity ligation assay and assessed the stability of Gli3stability in BrCa cell extracts by western blots. We also studied the effect of ER-α knockdown ordestabilization (by fulvestrant treatment) on Gli3 stability, formation of intranuclear ER-α-Gli3complexes and Gli reporter activity. We also measured the expression level of Gli target genes in thepresence and absence of estradiol by qPCR in BrCa cells. Lastly, we evaluated the importance of Gli3expression on BrCa growth by Gli3 knockdown and Cyquant assay. Results: We found that ER co-immunoprecipitates with Gli3. Transfection with ER-α increased Glireporter activity which was further increased by estradiol treatment. Acute (2hr) estradiol treatmentincreased intranuclear ER-α-Gli3 complex formation in BrCa cells. Chronic (48hr) estradiol treatmentincreased Gli3 stability and endogenous activity in BrCa cells. Destabilization or knockdown of ER-αdecreased estradiol-induced formation of ER-α-Gli3 complexes as well as Gli activity and stability in BrCacells. In addition, siRNA knockdown of Gli3 reduced growth in BrCa cells. Conclusion: Collectively our results uncovered a new role of the steroid receptors ER and AR inregulating Gli oncogenic transcriptional activity in BrCa and PCa, respectively. Citation Format: Shabnam Massah, Jane Foo, Na Li, Sarah Truong, Mannan Nouri, Lishi Xie, Gail Prins, Ralph Buttyan, Nada Lallous, Artem Cherkasov. Characterization of Gli activation by the estrogen receptor in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5283.

5057 Background: Activating AR mutations ensure a continued AR activation by non-androgen steroid ligands, e.g. progesterone and glucocorticoids. CYP11A1 is the only enzyme that catalyzes the conversion of cholesterol to pregnenolone, from which all steroid hormones (glucocorticoids, mineralocorticoids, and sex steroids) are subsequently derived. ODM-208, an oral, selective inhibitor of CYP11A1, is being evaluated for safety and efficacy as a treatment of mCRPC in the ongoing CYPIDES phase I/II trial in men previously treated with both novel hormonal therapies and taxanes (ClinicalTrials.gov identifier: NCT03436485). Preliminary phase 1 results were previously reported (Fizazi K et al., ASCO GU 2022). Here we confirm that the in-vitro sensitivity of common AR mutations to ODM-208 treatment is mirrored in patient response in CYPIDES phase 1. Methods: ODM-208 was administered at daily doses between 6-150 mg (phase 2 dose: 10 mg) with dexamethasone and fludrocortisone, resulting in maximal suppression in all measured steroids at all doses. AR ligand-binding domain (LBD) mutations (L702H, V716M, W742L, W742C, H875Y, F877L, T878A, T878S, M896T, M896V) were assessed using a BEAMing assay (Sysmex Inostics) from plasma circulating cell-free DNA (cfDNA) collected before the first dose of ODM-208. The activation of wild-type (wt) and LBD mutated AR with various ligands was also studied in vitro using a luciferase reporter assay in AR negative PC3 cells. Results: 17 of 44 patients had at least one AR activating mutation, the most frequent being L702H (n = 11), T878A (n = 10), and H875Y (n = 6). Eleven out of the 16 evaluable patients (68%) with an AR LBD mutation achieved ≥50% reduction in serum PSA compared with 2 out of the 24 evaluable patients (8%) without an AR LBD mutation (P

Lineage plasticity of prostate cancer is associated with resistance to androgen receptor (AR) pathway inhibition (ARPI) and supported by a reactive tumor microenvironment. Here we show that changes in chondroitin sulfate (CS), a major glycosaminoglycan component of the tumor cell glycocalyx and extracellular matrix, is AR-regulated and promotes the adaptive progression of castration-resistant prostate cancer (CRPC) after ARPI. AR directly represses transcription of the 4-O-sulfotransferase gene CHST11 under basal androgen conditions, maintaining steady-state CS in prostate adenocarcinomas. When AR signaling is inhibited by ARPI or lost during progression to non-AR-driven CRPC as a consequence of lineage plasticity, CHST11 expression is unleashed, leading to elevated 4-O-sulfated chondroitin levels. Inhibition of the tumor cell CS glycocalyx delays CRPC progression, and impairs growth and motility of prostate cancer after ARPI. Thus, a reactive CS glycocalyx supports adaptive survival and treatment resistance after ARPI, representing a therapeutic opportunity in patients with advanced prostate cancer.

Numerous cancers, including prostate cancer (PCa), are addicted to transcription programs driven by superenhancers (SEs). The transcription of genes at SEs is enabled by the formation of phase-separated condensates by transcription factors and co-activators with intrinsically disordered regions. The androgen receptor (AR), main oncogenic driver in PCa, contains large disordered regions and is co-recruited with the co-activator MED1 to SEs to promote oncogenic programs. In this work, we show that dynamic AR-rich, liquid-like foci form in PCa models upon androgen stimulation and correlate with AR transcriptional activity. The co-activator MED1 plays an essential role in the formation of AR foci while AR antagonists hinder their formation. These results suggest that enhanced compartmentalization of AR and co-activators at SEs may play an important role in the activation of oncogenic transcription programs in PCa. A better understanding of the assembly and the regulation of these AR-rich compartments may provide novel therapeutic options.

International audience; Resistance to androgen-receptor (AR) directed therapies is, among other factors, associated with Myc transcription factors that are involved in development and progression of many cancers. Overexpression of N-Myc protein in prostate cancer (PCa) leads to its transformation to advanced neuroendocrine prostate cancer (NEPC) that currently has no approved treatments. N-Myc has a short half-life but acts as an NEPC stimulator when it is stabilized by forming a protective complex with Aurora A kinase (AURKA). Therefore, dual-inhibition of N-Myc and AURKA would be an attractive therapeutic avenue for NEPC. Following our computer-aided drug discovery approach, compounds exhibiting potent N-Myc specific inhibition and strong anti-proliferative activity against several N-Myc driven cell lines, were identified. Thereafter, we have developed dual inhibitors of N-Myc and AURKA through structure-based drug design approach by merging our novel N-Myc specific chemical scaffolds with fragments of known AURKA inhibitors. Favorable binding modes of the designed compounds to both N-Myc and AURKA target sites have been predicted by docking. A promising lead compound, 70812, demonstrated low-micromolar potency against both N-Myc and AURKA in vitro assays and effectively suppressed NEPC cell growth.

Gain-of-function mutations in human androgen receptor (AR) are among the major causes of drug resistance in prostate cancer (PCa). Identifying mutations that cause resistant phenotype is of critical importance for guiding treatment protocols, as well as for designing drugs that do not elicit adverse responses. However, experimental characterization of these mutations is time consuming and costly, thus, predictive models are needed to anticipate resistant mutations and to guide the drug discovery process. In this work, we leverage experimental data collected on 68 AR mutants, either observed in the clinic or described in the literature, to train a deep neural network (DNN) that predicts the response of these mutants to currently used and experimental anti-androgens and testosterone. We demonstrate that the use of this DNN, with general 2D descriptors, provides a more accurate prediction of the biological outcome (inhibition, activation, no-response, mixed-response) in AR mutant-drug pairs compared to other machine learning approaches. Finally, the developed approach was used to make predictions of AR mutant response to the latest AR inhibitor darolutamide, which were then validated by in-vitro experiments.

Androgen receptor (AR) signalling is essential in nearly all prostate cancers. Any alterations to AR-mediated transcription can have a profound effect on carcinogenesis and tumor growth. While mutations of the AR protein have been extensively studied, little is known about those somatic mutations that occur at the non-coding regions where AR binds DNA. Using clinical whole genome sequencing, we show that AR binding sites have a dramatically increased rate of mutations that is greater than any other transcription factor and specific to only prostate cancer. Demonstrating this may be common to lineage-specific transcription factors, estrogen receptor binding sites were also found to have elevated rate of mutations in breast cancer. We provide evidence that these mutations at AR binding sites, and likely other related transcription factors, are caused by faulty repair of abasic sites. Overall, this work demonstrates that non-coding AR binding sites are frequently mutated in prostate cancer and can impact enhancer activity., Androgen receptor (AR) mediated transcription is critical to prostate tumorigenesis and development. Here, utilising clinical whole genome sequencing data, the authors show that the non-coding AR binding sites on DNA are frequently mutated in prostate cancer potentially due to faulty base excision repair mechanisms

We have explored the effects of 20(S)-protopanaxadiol (aPPD), a naturally derived ginsenoside, against androgen receptor (AR) positive castration resistant prostate cancer (CRPC) xenograft tumors and have examined its interactions with AR. In silico docking studies for aPPD binding to AR, alongside transactivation bioassays and in vivo efficacy studies were carried out in the castration-resistant C4-2 xenograft model. Immunohistochemical (IHC) and Western blot analyses followed by evaluation of AR, apoptotic, cell cycle and proliferative markers in excised tumors was performed. The growth of established CRPC tumors was inhibited by 53% with aPPD and a corresponding decrease in serum PSA was seen compared to controls. The IHC data revealed that Ki-67 was significantly lower for aPPD treated tumors and was associated with elevated p21 and cleaved caspase-3 expression, compared to vehicle treatment. Furthermore, aPPD decreased AR protein expression in xenograft tumors, while significantly upregulating p27 and Bax protein levels. In vitro data supporting this suggests that aPPD binds to and significantly inhibits the N-terminal or the DNA binding domains of AR. The AR androgen binding site docking score for androgen (dihydrotestosterone) was −11.1, while that of aPPD was −7.1. The novel findings described herein indicate aPPD potently inhibits PCa in vivo partly via inhibition of a site on the AR N-terminal domain. This manifested as cell cycle arrest and concurrent induction of apoptosis via an increase in Bax, cleaved-caspase-3, p27 and p21 expression.

Simple Summary Prostate cancer (PCa) is the most commonly diagnosed non-skin cancer in men and one of the leading causes of cancer-related death. The driver of PCa proliferation and growth is the androgen receptor (AR) and inhibiting this receptor is the standard of care for patients, following surgery or radiotherapy. Unfortunately, the effectiveness of current therapeutics is temporary, with the cancer eventually developing drug resistance. Among the mechanisms of resistance are the arising mutations in the AR that make the receptor promiscuously activated by drugs or non-specific ligands, thus promoting cancer progression. The aim of this study is to characterize the responses of 44 AR mutants, derived from PCa patients, to available steroids that activate the receptor as well as to various treatments currently used in the clinic. This work will help create a tool to guide the medical team in selecting the best personalized treatment option for each patient. Abstract Resistance to drug treatments is common in prostate cancer (PCa), and the gain-of-function mutations in human androgen receptor (AR) represent one of the most dominant drivers of progression to resistance to AR pathway inhibitors (ARPI). Previously, we evaluated the in vitro response of 24 AR mutations, identified in men with castration-resistant PCa, to five AR antagonists. In the current work, we evaluated 44 additional PCa-associated AR mutants, reported in the literature, and thus expanded the study of the effect of darolutamide to a total of 68 AR mutants. Unlike other AR antagonists, we demonstrate that darolutamide exhibits consistent efficiency against all characterized gain-of-function mutations in a full-length AR. Additionally, the response of the AR mutants to clinically used bicalutamide and enzalutamide, as well as to major endogenous steroids (DHT, estradiol, progesterone and hydrocortisone), was also investigated. As genomic profiling of PCa patients becomes increasingly feasible, the developed “AR functional encyclopedia” could provide decision-makers with a tool to guide the treatment choice for PCa patients based on their AR mutation status.

HSP27 is highly expressed in, and supports oncogene addiction of, many cancers. HSP27 phosphorylation is a limiting step for activation of this protein and a target for inhibition, but its highly disordered structure challenges rational structure-guided drug discovery. We performed multistep biochemical, structural, and computational experiments to define a spherical 24-monomer complex composed of 12 HSP27 dimers with a phosphorylation pocket flanked by serine residues between their N-terminal domains. Ivermectin directly binds this pocket to inhibit MAPKAP2-mediated HSP27 phosphorylation and depolymerization, thereby blocking HSP27-regulated survival signaling and client-oncoprotein interactions. Ivermectin potentiated activity of anti-androgen receptor and anti-EGFR drugs in prostate and EGFR/HER2-driven tumor models, respectively, identifying a repurposing approach for cotargeting stress-adaptive responses to overcome resistance to inhibitors of oncogenic pathway signaling.

While Myc is an essential regulator of growth in normal cells, it is also frequently associated with cancer progression, therapy-resistance and lethal outcomes in most human cancers. In prostate cancer (PCa), Myc transcription factors are implicated in the pathogenesis and progression of the full spectrum of PCa, from adenocarcinoma to advanced castration-resistant and neuroendocrine phenotypes. Though a high-value therapeutic target, clinically approved anti-Myc drugs have yet to be discovered. To elicit its oncogenic effects, Myc must form a heterodimer with its partner Max, which together bind DNA and activate transcription of a spectrum of target genes that promote cell growth, proliferation, metabolism, and apoptosis while blocking differentiation. In this study, we identified a binding site on the DNA-binding domain of the structurally ordered Myc-Max complex and employed a computer-aided rational drug discovery approach to identify small molecules that effectively inhibit Myc-Max functionality. A large-scale virtual screening protocol implementing structure-based methodologies was utilized to select a set of top-ranked compounds that were subsequently evaluated experimentally and characterized mechanistically for their ability to inhibit Myc-Max transcriptional activity and subsequent downstream functions, to reduce viability in PCa cell lines, disrupt protein-DNA interactions and to induce apoptosis as their mechanism of action. Among compounds identified that effectively inhibit Myc-Max activity with low to mid-micromolar range potency and no or minimal generic cytotoxicity, VPC-70067, a close analog of the previously identified Myc inhibitor 10058-F4, served as proof-of-concept that our in silico drug discovery strategy performed as expected. Compound VPC-70063, of a chemically different scaffold, was the best performer in a panel of in vitro assays, and the forerunner for future hit-to-lead optimization efforts. These findings lay a foundation for developing more potent, specific and clinically optimized Myc-Max inhibitors that may serve as promising therapeutics, alone or in combination with current anti-cancer treatments, for treatment of specific phenotypes or heterogeneous tumors.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a versatile RNA-binding protein playing a critical role in alternative pre-mRNA splicing regulation in cancer. Emerging data have implicated hnRNP A1 as a central player in a splicing regulatory circuit involving its direct transcriptional control by c-Myc oncoprotein and the production of the constitutively active ligand-independent alternative splice variant of androgen receptor, AR-V7, which promotes castration-resistant prostate cancer (CRPC). As there is an urgent need for effective CRPC drugs, targeting hnRNP A1 could, therefore, serve a dual purpose of preventing AR-V7 generation as well as reducing c-Myc transcriptional output. Herein, we report compound VPC-80051 as the first small molecule inhibitor of hnRNP A1 splicing activity discovered to date by using a computer-aided drug discovery approach. The inhibitor was developed to target the RNA-binding domain (RBD) of hnRNP A1. Further experimental evaluation demonstrated that VPC-80051 interacts directly with hnRNP A1 RBD and reduces AR-V7 messenger levels in 22Rv1 CRPC cell line. This study lays the groundwork for future structure-based development of more potent and selective small molecule inhibitors of hnRNP A1&ndash, RNA interactions aimed at altering the production of cancer-specific alternative splice isoforms.

International audience; The inhibition of the androgen receptor (AR) is an established strategy in prostate cancer (PCa) treatment until drug resistance develops either through mutations in the ligand-binding domain (LBD) portion of the receptor or its deletion. We previously identified a druggable pocket on the DNA binding domain (DBD) dimerization surface of the AR and reported several potent inhibitors that effectively disrupted DBD-DBD interactions and consequently demonstrated certain antineoplastic activity. Here we describe further development of small molecule inhibitors of AR DBD dimerization and provide their broad biological characterization. The developed compounds demonstrate improved activity in the mammalian two-hybrid assay, enhanced inhibition of AR-V7 transcriptional activity, and improved microsomal stability. These findings position us for the development of AR inhibitors with entirely novel mechanisms of action that would bypass most forms of PCa treatment resistance, including the truncation of the LBD of the AR.

Acquired resistance to a drug treatment is a common problem across many cancers including prostate cancer (PCa) - one of the major factors for male mortality. The androgen receptor (AR) continues to be the main therapeutic PCa target and despite the success of modern targeted therapies such as enzalutamide, resistance to these drugs eventually develops. The AR has found many ways to adapt to treatments including overexpression and production of functional, constitutively active splice variants. However, of particular importance are point mutations in the ligand binding domain of the protein that convert anti-androgens into potent AR agonists. This mechanism appears to be especially prevalent with the AR in spite of some distant similarities to other hormone nuclear receptors. Despite the AR being one of the most studied and attended targets in cancer, those gain-of-function mutations in the receptor remain a significant challenge for the development of PCa therapies. This drives the need to fully characterize such mutations and to consistently screen PCa patients for their occurrence to prevent adverse reactions to anti-androgen drugs. Novel treatments should also be developed to overcome this resistance mechanism and more attention should be given to the possibility of similar occurrences in other cancers.

Estrogen receptor-α positive (ERα+) breast cancers represent 75% of all invasive breast cancer cases, while de novo or acquired resistance to ER-directed therapy is also on the rise. Numerous factors contribute to this phenomenon including the recently-reported ESR1 gene mutations such as Y537S, which amplifies co-activator interactions with ERα and promotes constitutive activation of ERα function. Herein, we propose that direct targeting of the activation function-2 (AF2) site on ERα represents a promising alternative therapeutic strategy to overcome mutation-driven resistance in breast cancer. A systematic computer-guided drug discovery approach was employed to develop a potent ERα inhibitor that was extensively evaluated by a series of experiments to confirm its AF2-specific activity. We demonstrate that the developed small-molecule inhibitor effectively prevents ERα-coactivator interactions and exhibits a strong anti-proliferative effect against tamoxifen-resistant cells, as well as downregulates ERα-dependent genes and effectively diminishes the receptor binding to chromatin. Notably, the identified lead compound successfully inhibits known constitutively-active, resistance-associated mutant forms of ERα observed in clinical settings. Overall, this study reports the development of a novel class of ERα AF2 inhibitors, which have the potential to effectively inhibit ERα activity by a unique mechanism and to circumvent the issue of mutation-driven resistance in breast cancer.

Human androgen receptor (AR) is a hormone-activated transcription factor that is an important drug target in the treatment of prostate cancer. Current small-molecule AR antagonists, such as enzalutamide, compete with androgens that bind to the steroid-binding pocket of the AR ligand–binding domain (LBD). In castration-resistant prostate cancer (CRPC), drug resistance can manifest through AR-LBD mutations that convert AR antagonists into agonists, or by expression of AR variants lacking the LBD. Such treatment resistance underscores the importance of novel ways of targeting the AR. Previously, we reported the development of a series of small molecules that were rationally designed to selectively target the AR DNA-binding domain (DBD) and, hence, to directly interfere with AR–DNA interactions. In the current work, we have confirmed that the lead AR DBD inhibitor indeed directly interacts with the AR-DBD and tested that substance across multiple clinically relevant CRPC cell lines. We have also performed a series of experiments that revealed that genome-wide chromatin binding of AR was dramatically impacted by the lead compound (although with lesser effect on AR variants). Collectively, these observations confirm the novel mechanism of antiandrogen action of the developed AR-DBD inhibitors, establishing proof of principle for targeting DBDs of nuclear receptors in endocrine cancers. Mol Cancer Ther; 16(10); 2281–91. ©2017 AACR.

Summary There has been a resurgence of interest in the development of androgen receptor (AR) inhibitors with alternative modes of action to overcome the development of resistance to current therapies. We demonstrated previously that one promising strategy for combatting mutation-driven drug resistance is to target the Binding Function 3 (BF3) pocket of the receptor. Here we report the development of a potent BF3 inhibitor, 3-(2,3-dihydro-1 H -indol-2-yl)-1 H -indole, which demonstrates excellent antiandrogen potency and anti-PSA activity and abrogates the androgen-induced proliferation of androgen-sensitive (LNCaP) and enzalutamide-resistant (MR49F) PCa cell lines. Moreover, this compound effectively reduces the expression of AR-dependent genes in PCa cells and effectively inhibits tumor growth in vivo in both LNCaP and MR49F xenograft models. These findings provide evidence that targeting the AR BF3 pocket represents a viable therapeutic approach to treat patients with advanced and/or resistant prostate cancer.

SummaryUpregulation of CAD, the multifunctional protein that initiates and controls the de novo biosynthesis of pyrimidines in animals, is essential for cell proliferation. Deciphering the architecture and functioning of CAD is of interest for its potential usage as an antitumoral target. However, there is no detailed structural information about CAD other than that it self-assembles into hexamers of ∼1.5 MDa. Here we report the crystal structure and functional characterization of the dihydroorotase domain of human CAD. Contradicting all assumptions, the structure reveals an active site enclosed by a flexible loop with two Zn2+ ions bridged by a carboxylated lysine and a third Zn coordinating a rare histidinate ion. Site-directed mutagenesis and functional assays prove the involvement of the Zn and flexible loop in catalysis. Comparison with homologous bacterial enzymes supports a reclassification of the DHOase family and provides strong evidence against current models of the architecture of CAD.

The development of new antiandrogens, such as enzalutamide, or androgen synthesis inhibitors like abiraterone has improved patient outcomes in the treatment of advanced prostate cancer. However, due to the development of drug resistance and tumor cell survival, a majority of these patients progress to the refractory state of castration-resistant prostate cancer (CRPC). Thus, newer therapeutic agents and a better understanding of their mode of action are needed for treating these CRPC patients. We demonstrated previously that targeting the Binding Function 3 (BF3) pocket of the androgen receptor (AR) has great potential for treating patients with CRPC. Here, we explore the functional activity of this site by using an advanced BF3-specific small molecule (VPC-13566) that was previously reported to effectively inhibit AR transcriptional activity and to displace the BAG1L peptide from the BF3 pocket. We show that VPC-13566 inhibits the growth of various prostate cancer cell lines, including an enzalutamide-resistant cell line, and reduces the growth of AR-dependent prostate cancer xenograft tumors in mice. Importantly, we have used this AR-BF3 binder as a chemical probe and identified a co-chaperone, small glutamine-rich tetratricopeptide repeat (TPR)-containing protein alpha (SGTA), as an important AR-BF3 interacting partner. Furthermore, we used this AR-BF3–directed small molecule to demonstrate that inhibition of AR activity through the BF3 functionality can block translocation of the receptor into the nucleus. These findings suggest that targeting the BF3 site has potential clinical importance, especially in the treatment of CRPC and provide novel insights on the functional role of the BF3 pocket. Mol Cancer Ther; 15(12); 2936–45. ©2016 AACR.

The androgen receptor (AR) is an important regulator of genes responsible for the development and recurrence of prostate cancer. Current therapies for this disease rely on small-molecule inhibitors that block the transcriptional activity of the AR. Recently, major advances in the development of novel AR inhibitors resulted from X-ray crystallographic information on the receptor and utilization of in silico drug design synergized with rigorous experimental testing.Herein, we describe a drug-discovery pipeline for in silico screening for small molecules that target an allosteric region on the AR termed the binding-function 3 (BF3) site. Following the identification of potential candidates, the compounds are tested in cell culture and biochemical assays for their ability to interact with and inhibit the AR. The described pipeline is readily accessible and could be applied in drug design efforts toward any surface-exposed region on the AR or other related steroid nuclear receptor.

The androgen receptor (AR) is an important regulator of genes responsible for the development and recurrence of prostate cancer. Current therapies for this disease rely on small-molecule inhibitors that block the transcriptional activity of the AR. Recently, major advances in the development of novel AR inhibitors resulted from X-ray crystallographic information on the receptor and utilization of in silico drug design synergized with rigorous experimental testing.Herein, we describe a drug-discovery pipeline for in silico screening for small molecules that target an allosteric region on the AR termed the binding-function 3 (BF3) site. Following the identification of potential candidates, the compounds are tested in cell culture and biochemical assays for their ability to interact with and inhibit the AR. The described pipeline is readily accessible and could be applied in drug design efforts toward any surface-exposed region on the AR or other related steroid nuclear receptor.

Background The androgen receptor (AR) is a pivotal drug target for the treatment of prostate cancer, including its lethal castration-resistant (CRPC) form. All current non-steroidal AR antagonists, such as hydroxyflutamide, bicalutamide, and enzalutamide, target the androgen binding site of the receptor, competing with endogenous androgenic steroids. Several AR mutations in this binding site have been associated with poor prognosis and resistance to conventional prostate cancer drugs. In order to develop an effective CRPC therapy, it is crucial to understand the effects of these mutations on the functionality of the AR and its ability to interact with endogenous steroids and conventional AR inhibitors. Results We previously utilized circulating cell-free DNA (cfDNA) sequencing technology to examine the AR gene for the presence of mutations in CRPC patients. By modifying our sequencing and data analysis approaches, we identify four additional single AR mutations and five mutation combinations associated with CRPC. Importantly, we conduct experimental functionalization of all the AR mutations identified by the current and previous cfDNA sequencing to reveal novel gain-of-function scenarios. Finally, we evaluate the effect of a novel class of AR inhibitors targeting the binding function 3 (BF3) site on the activity of CRPC-associated AR mutants. Conclusions This work demonstrates the feasibility of a prognostic and/or diagnostic platform combining the direct identification of AR mutants from patients’ serum, and the functional characterization of these mutants in order to provide personalized recommendations regarding the best future therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0864-1) contains supplementary material, which is available to authorized users.

Recurrent, metastatic prostate cancer continues to be a leading cause of cancer-death in men. The androgen receptor (AR) is a modular, ligand-inducible transcription factor that regulates the expression of genes that can drive the progression of this disease, and as a consequence, this receptor is a key therapeutic target for controlling prostate cancer. The current drugs designed to directly inhibit the AR are called anti-androgens, and all act by competing with androgens for binding to the androgen/ligand binding site. Unfortunately, with the inevitable progression of the cancer to castration resistance, many of these drugs become ineffective. However, there are numerous other regulatory sites on this protein that have not been exploited therapeutically. The regulation of AR activity involves a cascade of complex interactions with numerous chaperones, co-factors and co-regulatory proteins, leading ultimately to direct binding of AR dimers to specific DNA androgen response elements within the promoter and enhancers of androgen-regulated genes. As part of the family of nuclear receptors, the AR is organized into modular structural and functional domains with specialized roles in facilitating their inter-molecular interactions. These regions of the AR present attractive, yet largely unexploited, drug target sites for reducing or eliminating androgen signaling in prostate cancers. The design of small molecule inhibitors targeting these specific AR domains is only now being realized and is the culmination of decades of work, including crystallographic and biochemistry approaches to map the shape and accessibility of the AR surfaces and cavities. Here, we review the structure of the AR protein and describe recent advancements in inhibiting its activity with small molecules specifically designed to target areas distinct from the receptor’s androgen binding site. It is anticipated that these new classes of anti-AR drugs will provide an additional arsenal to treat castration-resistant prostate cancer.

The androgen receptor (AR) is a hormone inducible transcription factor that continues to be an important drug-target to prevent or slow the progression of prostate cancer. Current small molecule inhibitors, such as Enzalutamide (anti-androgens), compete with naturally occurring steroids that bind to the hormone binding pocket of the AR ligand binding domain (LBD). In advanced or castration resistant prostate cancer (CRPC), mutations in the LBD confer drug-resistance by converting anti-androgens into agonists, prompting research to develop small molecule inhibitors that target different sites on the AR protein surface. Recently, we characterized a set of small molecules that could interact with the DNA binding domain (DBD) of the AR and block its transcriptional activity. Here, we extend the pioneering observations by clarifying the mechanism of two classes of compounds that either block AR-chromatin interactions or reduce AR-dimerization in the cell nucleus. Compound efficacy is demonstrated across multiple prostate cancer cells lines, including enzalutamide resistant forms, with respect to AR transactivation, cell viability and expression of downstream genes. We also characterize the pharmacological properties of the lead compound and its effects on tumour xenografts in mice. Collectively, these results lay the foundation for the development of alternative prostate cancer drugs that interfere with the biochemical function of the activated, nuclear localized AR. Citation Format: Kush Dalal, Aishwariya Sharma, Mani Roshan-Moniri, Hendrik Borgmann, Nada Lallous, Shannon Awrey, Huifang Li, Fuqiang Ban, Eric LeBlanc, Artem Cherkasov, Paul S. Rennie. Inhibition of the androgen receptor at two drug-targetable sites on the DNA-binding domain protein surface. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4644.

CAD is a 243 kDa eukaryotic multifunctional polypeptide that catalyzes the first three reactions ofde novopyrimidine biosynthesis: glutamine-dependentcarbamyl phosphate synthetase,aspartate transcarbamylase anddihydroorotase (DHO). In prokaryotes, these activities are associated with monofunctional proteins, for which crystal structures are available. However, there is no detailed structural information on the full-length CAD protein or any of its functional domains apart from that it associates to form a homohexamer of ∼1.5 MDa. Here, the expression, purification and crystallization of the DHO domain of human CAD are reported. The DHO domain forms homodimers in solution. Crystallization experiments yielded small crystals that were suitable for X-ray diffraction studies. A diffraction data set was collected to 1.75 Å resolution using synchrotron radiation at the SLS, Villigen, Switzerland. The crystals belonged to the orthorhombic space groupC2221, with unit-cell parametersa= 82.1,b= 159.3,c= 61.5 Å. The Matthews coefficient calculation suggested the presence of one protein molecule per asymmetric unit, with a solvent content of 48%.

International audience; Human UHRF1 belongs to the unique mammalian family of proteins which contain a SET- and RING finger-associated (SRA) domain. This 180-residue domain has been reported to play key roles in the functions of the protein. It allows UHRF1 to bind methylated DNA, histone deacetylase 1 and DNA methyltransferase 1, suggesting a bridge between DNA methylation and the histone code. No structural data is available for any SRA domain. Native and SeMet-labelled SRA domains of human UHRF1 were overexpressed in Escherichia coli cells, purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. A complete MAD data set was collected to 2.2 A resolution at 100 K. Crystals of the SeMet-labelled protein belonged to the trigonal space group P3(2)21, with unit-cell parameters a = b = 53.78, c = 162.05 A.

The mutation-driven transformation of clinically used anti-androgens into agonists of human androgen receptor (AR) represents a major challenge in the current treatment of prostate cancer (PCa). To address this problem, we have developed a novel class of AR inhibitors targeting the DNA-binding domain (DBD) of the receptor, which is distanced from the mutations-prone androgen binding site (ABS) in the ligand-binding domain (LBD) of the AR, targeted by all conventional antiandrogens. While many members of the developed phenyl-thiazol-2-yl-morpholine series demonstrated potent sub-micromolar inhibition of the wild-type AR, some compounds also exhibited an undesired partial agonistic effect toward the T877A mutated form of the receptor, implying their cross-interaction with the AR ABS. To study the molecular basis of the cross-reactivity, we have solved the T877A mutated form of the AR LBD in complex with one developed compound exhibiting such unwanted partial agonism. Based on the resolved crystal structure, we have identified critical protein-ligand interactions and conformational changes in wild-type and T877A forms of AR that drive observed agonistic effects. The identified structural basis has further been used to modify the scaffold of developed AR DBD binders to eliminate their cross-reactivity toward T877A mutated AR LBD. In particular, the replacement of the phenyl ring with less hydrophobic heterocycles resulted in a series of inhibitors that did not demonstrate affinity toward ABS or exhibit an undesired agonistic effect on AR. This study provides insights into the development of AR inhibitors with high specificity, which may help overcome the mutation-driven resistance of anti-AR drugs in the treatment of PCa. Citation Format: Huifang Li, Nada Lallous, Kush Dalal, Eric Leblanc, Fuqiang Ban, Fabrice Ciesielski, Paul S. Rennie, Artem Cherkasov. Structure-based study to overcome cross-reactivity of novel androgen receptor inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3653. doi:10.1158/1538-7445.AM2015-3653

Introduction Current approaches to inhibit oestrogen receptor-alpha (ERα) are focused on targeting its hormone-binding pocket and have limitations. Thus, we propose that inhibitors that bind to a coactivator-binding pocket on ERα, called activation function 2 (AF2), might overcome some of these limitations. Methods In silico virtual screening was used to identify small-molecule ERα AF2 inhibitors. These compounds were screened for inhibition of ERα transcriptional activity using stably transfected T47D-KBluc cell line. A direct physical interaction between the AF2 binders and the ERα protein was measured using biolayer interferometry (BLI) and an ERα coactivator displacement assay. Cell viability was assessed by MTS assay in ERα-positive MCF7 cells, tamoxifen-resistant (TamR) cell lines TamR3 and TamR6, and ERα-negative MDA-MB-453 and HeLa cell lines. In addition, ERα inhibition in TamR cells and the effect of compounds on mRNA and protein expression of oestrogen-dependent genes, pS2, cathepsin D and cell division cycle 2 (CDC2) were determined. Results Fifteen inhibitors from two chemical classes, derivatives of pyrazolidine-3,5-dione and carbohydrazide, were identified. In a series of in vitro assays, VPC-16230 of the carbohydrazide chemical class emerged as a lead ERα AF2 inhibitor that significantly downregulated ERα transcriptional activity (half-maximal inhibitory concentration = 5.81 μM). By directly binding to the ERα protein, as confirmed by BLI, VPC-16230 effectively displaced coactivator peptides from the AF2 pocket, confirming its site-specific action. VPC-16230 selectively suppressed the growth of ERα-positive breast cancer cells. Furthermore, it significantly inhibited ERα mediated transcription in TamR cells. More importantly, it reduced mRNA and protein levels of pS2, cathepsin D and CDC2, validating its ER-directed activity. Conclusion We identified VPC-16230 as an ERα AF2-specific inhibitor that demonstrated promising antiproliferative effects in breast cancer cell lines, including TamR cells. VPC-16230 reduced the expression of ERα-inducible genes, including CDC2, which is involved in cell division. We anticipate that the application of ERα AF2 inhibitors will provide a novel approach that can act as a complementary therapeutic to treat ERα-positive, tamoxifen-resistant and metastatic breast cancers. Electronic supplementary material The online version of this article (doi:10.1186/s13058-015-0529-8) contains supplementary material, which is available to authorized users.