Virtual screening and biological evaluation of PPARγ antagonists as potential anti-prostate cancer agents.

The peroxisome proliferator-activated receptor gamma (PPARγ) was identified as an oncogene and it plays a key role in prostate cancer (PC) development and progression. PPARγ antagonists have been shown to inhibit PC cell growth. Herein, we describe a virtual screening-based approach that led to the discovery of novel PPARγ antagonist chemotypes that bind at the allosteric pocket. Arg288, Lys367, and His449 appear to be important for PPARγ antagonist binding.

Blood-based gene expression signature associated with metastatic castrate-resistant prostate cancer patient response to abiraterone plus prednisone or enzalutamide.

BACKGROUND: Precision medicine approaches for managing patients with metastatic castrate-resistant prostate cancer (mCRPC) are lacking. Non-invasive approaches for molecular monitoring of disease are urgently needed, especially for patients suffering from bone metastases for whom tissue biopsy is challenging. Here we utilized baseline blood samples to identify mCRPC patients most likely to benefit from abiraterone plus prednisone (AAP) or enzalutamide. METHODS: Baseline blood samples were collected for circulating tumor cell (CTC) enumeration and qPCR-based gene expression analysis from 51 men with mCRPC beginning treatment with abiraterone or enzalutamide. RESULTS: Of 51 patients (median age 68 years [51-82]), 22 received AAP (abiraterone 1000 mg/day plus prednisone 10 mg/day) and 29 received enzalutamide (160 mg/day). The cohort was randomly divided into training (n = 37) and test (n = 14) sets. Baseline clinical variables (Gleason score, PSA, testosterone, and hemoglobin), CTC count, and qPCR-based gene expression data for 141 genes/isoforms in CTC-enriched blood were analyzed with respect to overall survival (OS). Genes with expression most associated with OS included MSLN, ARG2, FGF8, KLK3, ESRP2, NPR3, CCND1, and WNT5A. Using a Cox-elastic net model for our test set, the 8-gene expression signature had a c-index of 0.87 (95% CI [0.80, 0.94]) and was more strongly associated with OS than clinical variables or CTC count alone, or a combination of the three variables. For patients with a low-risk vs. high-risk gene expression signature, median OS was not reached vs. 18 months, respectively (HR 5.32 [1.91-14.80], p = 0.001). For the subset of 41 patients for whom progression-free survival (PFS) data was available, the median PFS for patients with a low-risk vs high-risk gene expression signature was 20 vs. 5 months, respectively (HR 2.95 [1.46-5.98], p = 0.003). CONCLUSIONS: If validated in a larger prospective study, this test may predict patients most likely to benefit from second-generation antiandrogen therapy.

Circulating tumor DNA analysis of metastatic renal cell carcinoma.

The genomic landscape of metastatic renal cell carcinoma (RCC) is not well understood, and currently available data suggest that it is functionally distinct from that of localized tumors. Additionally, the large number of approved and trial agents used to treat metastatic RCC likely cause selective adaptations in the tumors. Circulating tumor DNA (ctDNA) is a platform to non-invasively determine the genomic profiles of these tumors. The objectives of the present study were to corroborate previous ctDNA studies in metastatic RCC, to identify novel mutations in metastatic RCC, and to compare ctDNA profiles obtained from plasma and urine in patients with metastatic RCC. ctDNA sequencing using the plasma and urine of 50 patients with metastatic RCC who received ctDNA profiling as part of routine clinical care at a single institution was performed using an investigational 120-gene panel. Genomic alterations (GAs) were identified in all 50 patients. The genes with the most GAs were GNAS, PTEN, MYC, MET and HNF1A and novel mutations in additional genes were identified. A significant correlation between the number of GAs detected in matched urine and plasma samples was also identified, but only 28.1% of GAs detected in plasma samples were also detected in matched urine samples. The results of the present study were consistent with those of the largest previous study of ctDNA from patients with metastatic RCC and may help identify additional potential targets for the treatment of such patients.

Peroxisome proliferator-activated receptor gamma controls prostate cancer cell growth through AR-dependent and independent mechanisms.

BACKGROUND: Prostate cancer (PC) remains a leading cause of cancer mortality and the most successful chemopreventative and treatment strategies for PC come from targeting the androgen receptor (AR). Although AR plays a key role, it is likely that other molecular pathways also contribute to PC, making it essential to identify and develop drugs against novel targets. Recent studies have identified peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor that regulates fatty acid (FA) metabolism, as a novel target in PC, and suggest that inhibitors of PPARγ could be used to treat existing disease. We hypothesized that PPARγ acts through AR-dependent and independent mechanisms to control PC development and growth and that PPARγ inhibition is a viable PC treatment strategy. METHODS: Immunohistochemistry was used to determine expression of PPARÒ¯ in a cohort of patients with PC. Standard molecular techniques were used to investigate the PPARÒ¯ signaling in PC cells as well a xenograft mouse model to test PPARÒ¯ inhibition in vivo. Kaplan-Meier curves were created using cBioportal. RESULTS: We confirmed the expression of PPARÒ¯ in human PC. We then showed that small molecule inhibition of PPARγ decreases the growth of AR-positive and -negative PC cells in vitro and that T0070907, a potent PPARγ antagonist, significantly decreased the growth of human PC xenografts in nude mice. We found that PPARγ antagonists or small interfering RNA (siRNA) do not affect mitochondrial activity nor do they cause apoptosis; instead, they arrest the cell cycle. In AR-positive PC cells, antagonists and siRNAs reduce AR transcript and protein levels, which could contribute to growth inhibition. AR-independent effects on growth appear to be mediated by effects on FA metabolism as the specific FASN inhibitor, Fasnall, inhibited PC cell growth but did not have an additive effect when combined with PPARγ antagonists. Patients with increased PPARÒ¯ target gene expression, but not alterations in PPARÒ¯ itself, were found to have significantly worse overall survival. CONCLUSIONS: Having elucidated the direct cancer cell effects of PPARγ inhibition, our studies have helped to determine the role of PPARγ in PC growth, and support the hypothesis that PPARγ inhibition is an effective strategy for PC treatment.

Factors that influence the androgen receptor cistrome in benign and malignant prostate cells.

The androgen receptor (AR) plays key roles in the development of prostate tissue and the development and progression of prostate cancer (PC). AR guides cytodifferentiation and homeostasis in benign luminal epithelial cells; however, in PC, AR instead drives the uncontrolled proliferation of these cells. This 'AR malignancy shift' (AMS) is a central event in tumorigenesis. Using a ChIP-seq approach in primary human tissues, cell lines, and mouse models, we demonstrate that the AMS occurs in every sample analyzed, suggesting that it is necessary for PC development. Using molecular and genetic techniques, we demonstrate that forkhead box (FOX)A1, HOXB13, GATA2, and c-JUN are involved in the regulation of the AMS. AR-binding sites (ARBS) are enriched for FOX, HOX, and GATA motifs in PC cells but not for c-JUN motifs in benign cells. We show that the SPOP mutation commonly found in localized PCs can cause the AMS but is not transformative on its own and must be coupled to another mutation to transform cells. We show that the AMS occurs in mouse models of PC as well and that chronic low T, which is associated with increased PC risk and aggressiveness in humans, also causes the AMS in mice. We have discovered a previously unrecognized, fundamental tenet of PC, one which explains how and why AR signaling is different in cancer and benign cells. Our work has the potential to be used to stratify patients with localized PC for specific treatments. Furthermore, our work suggests that the AMS is a novel target for the treatment and/or prevention of PC.

Different roles of peroxisome proliferator-activated receptor gamma isoforms in prostate cancer.

Due to the increasing occurrence of and high costs associated with prostate cancer (PC), there is an urgent need to develop novel PC treatment and chemoprevention strategies. Although androgen receptor (AR) signaling is significant in the development and progression of PC, other molecular pathways contribute as well. Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) has recently been implicated as an oncogene in PC, which may influence both the development and metastatic progression of the cancer. There are two isoforms of PPARγ, with PPARγ2 having an additional 30 amino acids at the amino terminus. Here, we investigated the differential expression and function of these two isoforms in benign and cancerous prostate epithelial cells. The findings from our immunohistochemistry (IHC) and RNA in situ hybridization experiments suggest that although both isoforms are expressed in benign human prostate tissue, PPARγ1 predominates in PC tissue. Our results from PC cell line experiments suggest that PPARγ1 contributes to the proliferation of some PC cells and that PPARγ2 represses PC cell growth. Our findings also suggest that PPARγ1 increases the growth and possibly the transformation of otherwise benign prostate epithelial cells. These results help to establish different roles for PPARγ isoforms in prostate cells, and support the hypothesis that PPARγ1 acts as an oncogene and that PPARγ2 acts as a tumor suppressor in prostate cells.

Mechanistic Investigation of the Androgen Receptor DNA-Binding Domain Inhibitor Pyrvinium.

Pyrvinium was identified as the first small molecule inhibitor of the androgen receptor (AR) DNA-binding domain (DBD). It was also among the first small molecules shown to directly inhibit the activity of AR splice variants (ARVs), which has important clinical implications in the treatment of castration-resistant prostate cancer. Important questions about pyrvinium's mechanism of action remain. Here, we demonstrate through mutational analysis that amino acids 609 and 612 are important for pyrvinium action. Nuclear magnetic resonance demonstrates a specific interaction between a soluble pyrvinium derivative and the AR DBD homodimer-DNA complex. Chromatin immunoprecipitation and electrophoretic mobility shift assay experiments demonstrate that, despite an interaction with this complex, pyrvinium does not alter the DNA-binding kinetics in either assay. AR immunoprecipitation followed by mass spectrometry was used to identify proteins whose interaction with AR is altered by pyrvinium. Several splicing factors, including DDX17, had reduced interactions with AR in the presence of pyrvinium. RNA sequencing of prostate cancer cells treated with pyrvinium demonstrated changes in splicing, as well as in several other pathways. However, pyrvinium did not alter the levels of ARVs in several prostate cancer cell lines. Taken together, our new data pinpoint the direct interaction between pyrvinium and AR DBD and shed light on the mechanism by which it inhibits AR transcriptional activity.

Androgen Receptor and PI3K Pathway Activity in Ovarian Cancer.

We sought to evaluate androgen receptor (AR) and PI3K pathway activity in ovarian cancer cell lines and tissue and determine if either pathway was correlated with growth of ovarian cancers. AR expression and activity were quantified using immunohistochemistry (IHC) and RT-qPCR in six ovarian cancer cell lines and 51 tissue samples. Phospho-mTOR and AKT expression were quantified by IHC as well. Cell growth was assessed in the presence of AR modulating drugs and metformin. We found that despite robust AR expression and activity, no cell line was dependent on androgen for growth. However, metformin inhibited activity in five of the six cell lines. Patient tissues had large variation in AR expression and activity, as well as in expression of phospho-mTOR and AKT, but none of these variables correlated with progression-free survival (PFS). AR expression and activity did not predict the dependence of ovarian cancer cell lines on androgens for growth, and AR expression and activity did not correlate with PFS. This result suggests that AR expression as a criterion for patient selection for clinical trials evaluating molecules targeting AR may not predict response for ovarian cancer patients.

The androgen receptor malignancy shift in prostate cancer.

BACKGROUND: Androgens and the androgen receptor (AR) are necessary for the development, function, and homeostatic growth regulation of the prostate gland. However, once prostate cells are transformed, the AR is necessary for the proliferation and survival of the malignant cells. This change in AR function appears to occur in nearly every prostate cancer. We have termed this the AR malignancy shift. METHODS: In this review, we summarize the current knowledge of the AR malignancy shift, including the DNA-binding patterns that define the shift, the transcriptome changes associated with the shift, the putative drivers of the shift, and its clinical implications. RESULTS: In benign prostate epithelial cells, the AR primarily binds consensus AR binding sites. In carcinoma cells, the AR cistrome is dramatically altered, as the AR associates with FOXA1 and HOXB13 motifs, among others. This shift leads to the transcription of genes associated with a malignant phenotype. In model systems, some mutations commonly found in localized prostate cancer can alter the AR cistrome, consistent with the AR malignancy shift. Current evidence suggests that the AR malignancy shift is necessary but not sufficient for transformation of prostate epithelial cells. CONCLUSIONS: Reinterpretation of prostate cancer genomic classification systems in light of the AR malignancy shift may improve our ability to predict clinical outcomes and treat patients appropriately. Identifying and targeting the molecular factors that contribute to the AR malignancy shift is not trivial but by doing so, we may be able to develop new strategies for the treatment or prevention of prostate cancer.

Synaptophysin expression on circulating tumor cells in patients with castration resistant prostate cancer undergoing treatment with abiraterone acetate or enzalutamide.

BACKGROUND: With the advent of secondary androgen receptor (AR)-targeted therapies in metastatic castration resistant prostate cancer (PC), nonadenocarcinoma PCs are becoming more prevalent. Many of these cancers express neuroendocrine markers, which may provide biomarkers for emergence of this disease state. We aimed to quantify the expression of synaptophysin (Syp) on circulating tumor cells (CTCs) from serial samples of patients being treated with abiraterone acetate or enzalutamide. METHODS: CTCs were isolated from 44 patients with castration resistant PC before starting abiraterone or enzalutamide, at 4, 8, and 12 weeks on therapy, and at progression. Patients were stratified into 3 groups: de novo resistance, short response, and long response. CTCs were enumerated on the CellSearch platform and Syp expression was quantified using the open fluorescent channel on the platform. Correlative analyses were performed. RESULTS: A baseline CTC count of 5 or greater was associated with a more rapid time to progression and increasing CTC counts correlated with emergence of drug resistance. Syp was readily detectable on the surface of CTCs, and baseline percentage CTC Syp expression was significantly associated with time to progression. Furthermore, in evaluable patients, percent CTC Syp expression increased with the emergence of drug resistance. We also found that prior exposure to AR-targeted therapies was inversely associated with progression free survival. CONCLUSIONS: We have demonstrated that Syp can be quantified on CTCs and that Syp expression correlates with resistance to abiraterone and enzalutamide. Larger studies testing Syp as a biomarker of emergence of nonadenocarcinoma disease and as a marker of response to AR-targeted therapies are warranted.

Identification of mechanisms of resistance to treatment with abiraterone acetate or enzalutamide in patients with castration-resistant prostate cancer (CRPC).

BACKGROUND: Two androgen receptor (AR)-targeted therapies, enzalutamide and abiraterone acetate plus prednisone (abiraterone), have been approved for the treatment of metastatic castration-resistant prostate cancer (CRPC). Many patients respond to these agents, but both de novo and acquired resistance are common. The authors characterized resistant phenotypes that emerge after treatment with abiraterone or enzalutamide. METHODS: Patients who received abiraterone or enzalutamide in the course of routine clinical care were consented for serial blood collection. A proprietary system (CellSearch) was used to enumerate and enrich circulating tumor cells (CTCs). RNA-sequencing (RNA-seq) was performed on pools of up to 10 epithelial cell adhesion molecule (EpCAM)-positive/CD45-negative CTCs. The impact of gene expression changes observed in CTCs between patients who responded or were resistant to abiraterone/enzalutamide therapies was further explored in a model cell line system. RESULTS: RNA-seq data from CTCs identified mutations commonly associated with CRPC as well as novel mutations, including several in the ligand-binding domain of AR that could facilitate escape from AR-targeted agents. Ingenuity pathway analysis of differentially regulated genes identified the transforming growth factor ß (TGFß) and cyclin D1 (CCND1) signaling pathways as significantly upregulated in drug-resistant CTCs. Transfection experiments using enzalutamide-sensitive and enzalutamide-resistant LNCaP cells confirmed the involvement of SMAD family member 3, a key mediator of the TGFß pathway, and of CCND1 in resistance to enzalutamide treatment. CONCLUSIONS: The current results indicate that RNA-seq of CTCs representing abiraterone and enzalutamide sensitive and resistant states can identify potential mechanisms of resistance. Therapies targeting the downstream signaling mediated by SMAD family member 3 (SMAD3) and CCND1, such as cyclin-dependent kinase 4/cyclin-dependent kinase 6 inhibitors, could provide new therapeutic options for the treatment of antiandrogen-resistant disease. Cancer 2018;124:1216-24. © 2017 American Cancer Society.

The role of peroxisome proliferator-activated receptor gamma in prostate cancer.

Despite great progress in the detection and treatment of prostate cancer, this disease remains an incredible health and economic burden. Although androgen receptor (AR) signaling plays a key role in the development and progression of prostate cancer, aberrations in other molecular pathways also contribute to the disease, making it essential to identify and develop drugs against novel targets, both for the prevention and treatment of prostate cancer. One promising target is the peroxisome proliferator-activated receptor gamma (PPARγ) protein. PPARγ was originally thought to act as a tumor suppressor in prostate cells because agonist ligands inhibited the growth of prostate cancer cells; however, additional studies found that PPARγ agonists inhibit cell growth independent of PPARγ. Furthermore, PPARγ expression increases with cancer grade/stage, which would suggest that it is not a tumor suppressor but instead that PPARγ activity may play a role in prostate cancer development and/or progression. Indeed, two new studies, taking vastly different, unbiased approaches, have identified PPARγ as a target in prostate cancer and suggest that PPARγ inhibition might be useful in prostate cancer prevention and treatment. These findings could lead to a new therapeutic weapon in the fight against prostate cancer.

Identification of neuron selective androgen receptor inhibitors.

AIM: To identify neuron-selective androgen receptor (AR) signaling inhibitors, which could be useful in the treatment of spinal and bulbar muscular atrophy (SBMA), or Kennedy's disease, a neuromuscular disorder in which deterioration of motor neurons leads to progressive muscle weakness. METHODS: Cell lines representing prostate, kidney, neuron, adipose, and muscle tissue were developed that stably expressed the CFP-AR-YFP FRET reporter. We used these cells to screen a library of small molecules for cell type-selective AR inhibitors. Secondary screening in luciferase assays was used to identify the best cell-type specific AR inhibitors. The mechanism of action of a neuron-selective AR inhibitor was examined in vitro using luciferase reporter assays, immunofluorescence microscopy, and immunoprecipitations. Rats were treated with the most potent compound and tissue-selective AR inhibition was examined using RT-qPCR of AR-regulated genes and immunohistochemistry. RESULTS: We identified the thiazole class of antibiotics as compounds able to inhibit AR signaling in a neuronal cell line but not a muscle cell line. One of these antibiotics, thiostrepton is able to inhibit the activity of both wild type and polyglutamine expanded AR in neuronal GT1-7 cells with nanomolar potency. The thiazole antibiotics are known to inhibit FOXM1 activity and accordingly, a novel FOXM1 inhibitor FDI-6 also inhibited AR activity in a neuron-selective fashion. The selective inhibition of AR is likely indirect as the varied structures of these compounds would not suggest that they are competitive antagonists. Indeed, we found that FOXM1 expression correlates with cell-type selectivity, FOXM1 co-localizes with AR in the nucleus, and that shRNA-mediated knock down of FOXM1 reduces AR activity and thiostrepton sensitivity in a neuronal cell line. Thiostrepton treatment reduces FOXM1 levels and the nuclear localization of beta-catenin, a known co-activator of both FOXM1 and AR, and reduces the association between beta-catenin and AR. Treatment of rats with thiostrepton demonstrated AR signaling inhibition in neurons, but not muscles. CONCLUSION: Our results suggest that thiazole antibiotics, or other inhibitors of the AR-FOXM1 axis, can inhibit AR signaling selectively in motor neurons and may be useful in the treatment or prevention of SBMA symptoms.

Vitamin K epoxide reductase regulation of androgen receptor activity.

Long-term use of warfarin has been shown to be associated with a reduced risk of prostate cancer. Warfarin belongs to the vitamin K antagonist class of anticoagulants, which inhibit vitamin K epoxide reductase (VKOR). The vitamin K cycle is primarily known for its role in γ-carboxylation, a rare post-translational modification important in blood coagulation. Here we show that warfarin inhibits the transcriptional activity of the androgen receptor (AR), an important driver of prostate cancer development and progression. Warfarin treatment or knockdown of its target VKOR inhibits the activity of AR both in cell lines and in mouse prostate tissue. We demonstrate that AR can be γ-carboxylated, and mapped the γ-carboxylation to glutamate residue 2 (E2) using mass spectrometry. However, mutation of E2 and other glutamates on AR failed to suppress the effects of warfarin on AR suggesting that inhibition of AR is γ-carboxylation independent. To identify pathways upstream of AR signaling that are affected by warfarin, we performed RNA-seq on prostates of warfarin-treated mice. We found that warfarin inhibited peroxisome proliferator-activated receptor gamma (PPARγ) signaling, which in turn, inhibited AR signaling. Although warfarin is unfit for use as a chemopreventative due to its anticoagulatory effects, our data suggest that its ability to reduce prostate cancer risk is independent of its anticoagulation properties. Furthermore, our data show that warfarin inhibits PPARγ and AR signaling, which suggests that inhibition of these pathways could be used to reduce the risk of developing prostate cancer.

Low Testosterone Alters the Activity of Mouse Prostate Stem Cells.

BACKGROUND: Low serum testosterone (low T) has been repeatedly linked to worse outcomes in men with newly diagnosed prostate cancer (PC). How low T contributes to these outcomes is unknown. Here we demonstrate that exposure to low T causes significant changes in the mouse prostate and prostate stem cells. METHODS: Mice were castrated and implanted with capsules to achieve castrate, normal, or sub-physiological levels of T. After 6 weeks of treatment, LC-MS/MS was used to quantify the levels of T and dihydrotestosterone (DHT) in serum and prostate tissue. FACS was used to quantify the percentages of purported prostate stem and transit amplifying (TA) cells in mouse prostates. Prostate tissues were also stained for the presence of CD68+ cells and RNA was extracted from prostate tissue or specific cell populations to measure changes in transcript levels with low T treatment. RESULTS: Despite having significantly different levels of T and DHT in the serum, T and DHT concentrations in prostate tissue from different T treatment groups were similar. Low T treatment resulted in significant alterations in the expression of androgen biosynthesis genes, which may be related to maintaining prostate androgen levels. Furthermore, the expression of androgen-regulated genes in the prostate was similar among all T treatment groups, demonstrating that the mouse prostate can maintain functional levels of androgens despite low serum T levels. Low T increased the frequency of prostate stem and TA cells in adult prostate tissue and caused major transcriptional changes in those cells. Gene ontology analysis suggested that low T caused inflammatory responses and immunofluorescent staining indicated that low T treatment led to the increased presence of CD68+ macrophages in prostate tissue. CONCLUSIONS: Low T alters the AR signaling axis which likely leads to maintenance of functional levels of prostate androgens. Low T also induces quantitative and qualitative changes in prostate stem cells which appear to lead to inflammatory macrophage infiltration. These changes are proposed to lead to an aggressive phenotype once cancers develop and may contribute to the poor outcomes in men with low T. Prostate 77:530-541, 2017. © 2016 Wiley Periodicals, Inc.

Vitamin K epoxide reductase expression and prostate cancer risk.

PURPOSE: Increasing evidence has demonstrated that men taking the anticoagulant warfarin have a lower risk of developing prostate cancer. This phenomenon is not observed in other cancers. We sought to determine if the target of warfarin, vitamin K epoxide reductase (VKOR), is expressed in benign and cancerous prostate tissues and if a functional single nucleotide polymorphism (SNP) in the VKOR gene is associated with prostate cancer risk. MATERIALS AND METHODS: The expression of VKOR was quantified by immunohistochemistry in an institutional series of 54 radical prostatectomy samples and metastatic biopsies, as well as in 40 other cancers and matched benign tissues on a tissue microarray. Genotyping of SNP rs2359612 was performed in a prospective series of 57 patients. RESULTS: VKOR is highly expressed in benign human prostate epithelial cells but is not expressed or expressed at very low levels in cancerous cells. This expression pattern is unique to prostate cancer. Additionally, the proportion of the carrier C allele of rs2359612 in the patients with prostate cancer was significantly higher than in the population, suggesting an association between this allele and the risk of having a diagnosis of prostate cancer. CONCLUSIONS: The expression of VKOR in benign prostate epithelial cells, along with the association between a functional VKOR SNP and prostate cancer risk, suggests a possible role for VKOR in mediating the effect of warfarin on prostate cancer risk. Larger multi-institutional cohort studies are warranted, as are molecular studies on the role of VKOR in prostate cancer development.

Revisiting the SAR of the Antischistosomal Aryl Hydantoin (Ro 13-3978).

The aryl hydantoin 1 (Ro 13-3978) was identified in the early 1980s as a promising antischistosomal lead compound. However, this series of aryl hydantoins produced antiandrogenic side effects in the host, a not unexpected outcome given their close structural similarity to the antiandrogenic drug nilutamide. Building on the known SAR of this compound series, we now describe a number of analogs of 1 designed to maximize structural diversity guided by incorporation of substructures and functional groups known to diminish ligand-androgen receptor interactions. These analogs had calculated polar surface area (PSA), measured LogD7.4, aqueous kinetic solubility, and estimated plasma protein binding values in ranges predictive of good ADME profiles. The principal SAR insight was that the hydantoin core of 1 is required for high antischistosomal activity. We identified several compounds with high antischistosomal efficacy that were less antiandrogenic than 1. These data provide direction for the ongoing optimization of antischistosomal hydantoins.

High-Throughput Screen for Inhibitors of Androgen Receptor-RUNX2 Transcriptional Regulation in Prostate Cancer.

Runt-related transcription factor 2 (RUNX2) plays a critical role in prostate cancer progression. RUNX2 interacts with the androgen receptor (AR) and modulates its transcriptional activity in a locus-specific manner. RUNX2 and AR synergistically stimulate a subset of genes, including the pro-oncogene snail family zinc finger 2 (SNAI2). AR-RUNX2 signaling cooperatively induces invasiveness of prostate cancer cells via SNAI2; and coexpression of AR, RUNX2, and SNAI2 in prostate cancer biopsy samples predicts disease recurrence. Competitive inhibition of AR alone could not disrupt the synergistic activation of SNAI2. We therefore established a phenotypic cell-based screening assay for compounds that could inhibit AR-RUNX2 synergistic activity either directly or indirectly. This assay was used to screen 880 compounds as a proof of concept, resulting in identification of several compounds that disrupted the synergistic stimulation of genes. Further investigation suggested the involvement of epidermal growth factor receptor (EGFR) signaling in AR/RUNX2 synergistic activity. Our assay is amenable to high-throughput screening and can be used to identify inhibitors of the AR-RUNX2 interaction in prostate cancer cells.