Androgen Receptor Binding Affinity Research Articles

Quantitative structure activity relationship studies of androgen receptor binding affinity of endocrine disruptor chemicals with index of ideality of correlation, their molecular docking, molecular dynamics and ADME studies

Endocrine disrupter chemicals (EDCs) are both natural and man-made chemicals that mimic, block or interfere with human hormonal system. In the present manuscript, QSAR modeling was performed for the androgen disruptors that interfere with biosynthesis, metabolism or action of androgens that causes adverse effects on male reproductive system. A set of 96 EDCs that exhibited affinity towards androgen receptors (Log RBA) in rats were employed for carrying out QSAR studies using Hybrid descriptors (combination of HFG and SMILES) through Monte Carlo Optimization. Using index of ideality of correlation (TF2), five splits were formed and predictability of five models resulting from these splits was assessed by various validation parameters. Models resulted from first split was the top most one with R2validation = 0.7878. Structural attributes responsible for change in endpoint were studied by employing correlation weights of structural attributes. In order to further validate the model, new EDCs were designed using these attributes. In silico molecular modelling studies were performed to assess the detailed interactions with the receptor. The binding energies of all the designed compounds were observed to be better than lead and are in the range of −10.46 to −14.80. Molecular dynamics simulation of 100 ns was performed for ED01 and NED05. The results revealed that the protein-ligand complex bearing NED05 was more stable than lead ED01 exhibiting better interactions with the receptor. Further, in an attempt to assess their metabolism, ADME studies were evaluated using SwissADME. The developed model enables to predict the characteristics of designed compounds in an authentic way.Communicated by Ramaswamy H. Sarma

Quick and efficient quantitative predictions of androgen receptor binding affinity for screening Endocrine Disruptor Chemicals using 2D-QSAR and Chemical Read-Across

Endocrine Disruptor Chemicals are synthetic or natural molecules in the environment that promote adverse modifications of endogenous hormone regulation in humans and/or in animals. In the present research, we have applied two-dimensional quantitative structure-activity relationship (2D-QSAR) modeling to analyze the structural features of these chemicals responsible for binding to the androgen receptors (logRBA) in rats. We have collected the receptor binding data from the EDKB database (https://www.fda.gov/science-research/endocrine-disruptor-knowledge-base/accessing-edkb-database) and then employed the DTC-QSAR tool, available from https://dtclab.webs.com/software-tools, for dataset division, feature selection, and model development. The final partial least squares model was evaluated using various stringent validation criteria. From the model, we interpreted that hydrophobicity, steroidal nucleus, bulkiness and a hydrogen bond donor at an appropriate position contribute to the receptor binding affinity, while presence of electron rich features like aromaticity and polar groups decrease the receptor binding affinity. Additionally we have also performed chemical Read-Across predictions using Read-Across-v3.1 available from https://sites.google.com/jadavpuruniversity.in/dtc-lab-software/home, and the results for the external validation metrics were found to be better than the QSAR-derived predictions. The best quality of external predictions emerged from the q-RASAR approach which combines both read-across and QSAR. To explore the essential features responsible for the receptor binding, pharmacophore mapping, molecular docking along with molecular dynamics simulation were also performed, and the results are in accordance with the QSAR/q-RASAR findings.

Synthesis, bioactivity, and molecular docking of novel arylpiperazine derivatives as potential AR antagonists.

Prostate cancer is one of the malignant tumors and the second most common malignant tumor in men. Clinically used androgen receptor (AR)–targeted drugs can antagonize androgen and inhibit tumor growth, but these drugs can cause serious resistance problems. To develop novel AR antagonists, 22 kinds of arylpiperazine derivatives were designed and synthesized, and the derivatives 5, 8, 12, 19, 21, 22, 25, and 26 not only showed strong antagonistic potency (>55% inhibition) and binding affinities (IC50 <3 μM) to AR, but also showed stronger inhibitory activity to LNCaP cells versus PC-3 cells. Among them, derivative 21 exhibited the highest binding affinity for AR (IC50 = 0.65 μM) and the highest antagonistic potency (76.2% inhibition). Docking studies suggested that the derivative 21 is primarily bound to the AR-LBP site by the hydrophobic interactions. Overall, those results provided experimental methods for developing novel arylpiperazine derivatives as potent AR antagonists.

First report of q-RASAR modeling toward an approach of easy interpretability and efficient transferability.

Quantitative structure-activity relationship (QSAR) and read-across techniques have recently been merged into a new emerging field of read-across structure-activity relationship (RASAR) that uses the chemical similarity concepts of read-across (an unsupervised step) and finally develops a supervised learning model (like QSAR). The RASAR method has so far been used only in case of graded predictions or classification modeling. In this work, we attempt, for the first time, to apply RASAR for quantitative predictions (q-RASAR) using a case study of androgen receptor binding affinity data. We have computed a number of error-based and similarity-based measures such as weighted standard deviation of the predicted values, coefficient of variation of the computed predictions, average similarity level of close training compounds for each query molecule, standard deviation and coefficient of variation of similarity levels, maximum similarity levels to positive and negative close training compounds, a concordance measure indicating similarity to positive, negative or both classes of close training compounds, etc. We have clubbed these additional measures along with the selected chemical descriptors from the previously developed QSAR model and redeveloped new partial least squares models from the training set, and predicted the endpoint using the query data set. Interestingly, these new models outperform the internal and external validation quality of the original QSAR model. In this study, we have also introduced a new similarity-based concordance measure (Banerjee-Roy coefficient) that can significantly contribute to the model quality. A q-RASAR model also has the advantage over read-across predictions in providing easy interpretation and indicating quantitative contributions of important chemical features. The strategy described here should be applicable to other biological/toxicological/property data modeling for enhanced quality of predictions, easy interpretability, and efficient transferability.

Safety effect of fermented oyster extract on the endocrine disruptor assay in vitro and in vivo

Oyster (Crassostrea gigas) is a marine bivalve mollusk widely distributed in coastal areas, and have been long widely used in industrial resources. Several studies demonstrated that fermented oyster (FO) extract attribute to bone health, but whether administration of FO play as an endocrine disruptor has not been studied. Therefore, in the present study, we investigated the effect of FO on the endocrine system in vitro and in vivo. As the results of the competitive estrogen receptor (ER) and androgen receptor (AR) binding affinities, FO was not combined with ER-α, ER-β, and AR. However, 17β-estradiol and testosterone, used as positive control, were interacted with ER and AR, respectively. Meanwhile, oral administration of 100 mg/kg and 200 mg/kg of FO doesn’t have any harmful effect on the body weight, androgen-dependent sex accessory organs, estrogen-dependent-sex accessory organs, kidney, and liver in immature rats. In addition, FO supplementation has no effect on the serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, and 17β-estradiol. However, the relative weight of androgen- and estrogen-dependent organs were significantly increased by subcutaneously injection of 4.0 mg/kg of testosterone propionate (TP) and by orally administration of 1.0 μg of 17α-ethynyl estradiol (EE) in immature male and female rats, respectively. Furthermore, TP and EE administration markedly decreased the serum LH and FSH levels, which are similar those of mature Sprague-Dawley (SD) rat. Furthermore, the testosterone and 17β-estradiol levels were significantly enhanced in TP and EE-treated immature rats. Taken together, our findings showed that FO does not interact with ER and AR, suggesting consequentially FO does not play as a ligand for ER and AR. Furthermore, oral administration of FO did not act as an endocrine disruptor including androgenic activity, estrogenic activity, and abnormal levels of sex hormone, indicating FO may ensure the safety on endocrine system to develop dietary supplement for bone health.

Designed, synthesized and biological evaluation of proteolysis targeting chimeras (PROTACs) as AR degraders for prostate cancer treatment

As a continuation of our research on developing potent and potentially safe androgen receptor (AR) degrader, a series of novel proteolysis targeting chimeras (PROTACs) containing the phthalimide degrons with different linker were designed, synthesized and evaluated for their AR degradation activity against LNCaP (AR+) cell line. Most of the synthesized compounds displayed moderate to satisfactory AR binding affinity and might lead to antagonist activity against AR. Among them, compound A16 exhibited the best AR binding affinity (85%) and degradation activity against AR. Due to the strong fluorescence properties of pomalidomide derivatives, B10 was found to be effectively internalized and visualized in LNCaP (AR + ) cells than PC-3 (AR-) cells. Moreover, the molecular docking of A16 with AR and the active site of DDB1-CRBN E3 ubiquitin ligase complex provides guidance to design new PROTAC degrons targeting AR for prostate cancer therapy. These results represent a step toward the development of novel and improved AR PROTACs.

Endocrine disrupting potential of veterinary drugs by in vitro stably transfected human androgen receptor transcriptional activation assays

We describe the androgen receptor (AR) agonistic/antagonistic effects of 140 veterinary drugs regulated in Republic of Korea, by setting maximum residue limits. It was conducted using two in vitro test guidelines of the Organization for Economic Cooperation and Development (OECD)—the AR-EcoScreen AR transactivation (TA) assay and the 22Rv1/MMTV_GR-KO AR TA assay. These were performed alongside the AR binding affinity assay to confirm whether their AR agonistic/antagonistic effects are based on the binding affinity to AR. Prior to conducting the AR TA assay, the proficiency test was passed the proficiency performance criterion for the AR agonist and AR antagonist assays. Among the veterinary drugs tested, four veterinary drugs (dexamethasone, trenbolone, altrenogest, and nandrolone) and six veterinary drugs (cymiazole, dexamethasone, zeranol, phenothiazine, bromopropylate, and isoeugenol) were determined as AR agonist and AR antagonist, respectively in both in vitro AR TA assays. Zeranol exhibited weak AR agonistic effects with a PC10 value only in the 22Rv1/MMTV_GR-KO AR TA assay. Regarding changing the AR agonistic/antagonistic effects through metabolism, the AR antagonistic activities of zeranol, phenothiazine, and isoeugenol decreased significantly in the presence of phase I + II enzymes.These data indicate that various veterinary drugs could have the potential to disrupt AR-mediated human endocrine system. Furthermore, this is the first report providing information on AR agonistic/antagonistic effects of veterinary drugs using in vitro OECD AR TA assays.

Structure-based discovery of the endocrine disrupting effects of hydraulic fracturing chemicals as novel androgen receptor antagonists

Hydraulic fracturing (HF) technology is increasingly utilized for oil and gas extraction operations. The widespread use of HF has led to concerns of negative impacts on both the environment and human health. Indeed, the potential endocrine disrupting impacts of HF chemicals is one such knowledge gap. Herein, we used structure-based molecular docking to assess the binding affinities of 60 HF chemicals to the human androgen receptor (AR). Five HF chemicals had relatively high predicted AR binding affinity, suggesting the potential for endocrine disruption. We next assessed androgenic and antiandrogenic activities of these chemicals in vitro. Of the five candidate AR ligands, only Genapol®X–100 significantly modified AR transactivation. To better understand the structural effect of Genapol®X–100 on the potency of AR inhibition, we compared the antiandrogenic activity of Genapol®X–100 with that of its structurally similar chemical, Genapol®X–080. Interestingly, both Genapol®X–100 and Genapol®X–080 elicited an antagonistic effect at AR with 20% relative inhibitory concentrations of 0.43 and 0.89 μM, respectively. Furthermore, we investigated the mechanism of AR inhibition of these two chemicals in vitro, and found that both Genapol®X–100 and Genapol®X–080 inhibited AR through a noncompetitive mechanism. The effect of these two chemicals on the expression of AR responsive genes, e.g. PSA, KLK2, and AR, was also investigated. Genapol®X–100 and Genapol®X–080 altered the expression of these genes. Our findings heighten awareness of endocrine disruption by HF chemicals and provide evidence that noncompetitive antiandrogenic Genapol®X–100 could cause adverse endocrine health effects.

SAT-722 Structure-Based Discovery of Hydraulic Fracturing Chemicals as Novel Androgen Receptor Antagonists

Hydraulic fracturing (HF) technology is increasingly utilized for oil and gas extraction operations. The widespread use of HF has led to concerns of potential negative impacts on both the environment and human health. Indeed, the potential endocrine disrupting impacts of HF chemicals is one such knowledge gap. Herein, we used structure-based molecular docking to assess the binding affinities of 60 HF chemicals used in California to the human androgen receptor (AR). Five HF chemicals had relatively high AR binding affinity, suggesting the potential to disrupt AR effects. We next assessed androgenic and antiandrogenic activities of these chemicals in vitro. Of the five candidate AR ligands, only Genapol® X–100 was found to significantly reduce the AR transactivation by 22%. To better understand the structural effect of Genapol® X–100 on the potency of receptor inhibition, we compared the antiandrogenic activity of Genapol® X–100 with that of its structurally similar chemical, Genapol® X–080. Interestingly, both Genapol® X–100 and Genapol® X–080 elicited a significant antagonistic effect with 20% relative inhibitory concentrations (RIC20) of 0.43 and 0.89 μM, respectively. This indicated that Genapol® X–100 was more potent in inhibiting AR than Genapol® X–080, consistent with longer Genapol® X–100 chain length causing greater potency of AR activity inhibition. Furthermore, we investigated the mechanism of AR inhibition of these two chemicals in vitro. The result revealed that both Genapol® X–100 and Genapol® X–080 inhibited AR through noncompetitive binding mechanism. The effects of these two chemicals on the expression of AR responsive genes such as PSA, KLK2, and AR were also investigated. Genapol® X–100 and Genapol® X–080 notably altered the expression of these genes at relatively low concentrations of 0.5 µM to 1 µM. Using these integrated in vitro and in silico approaches, we identified HF chemicals as novel noncompetitive AR antagonists. Our findings heighten awareness of endocrine disruption by HF chemicals and provide evidence that noncompetitive antiandrogenic Genapol® X–100 could possibly cause adverse endocrine health effects in humans.

Synthesis, biological evaluation and molecular docking of 4-Amino-2H-benzo[h]chromen-2-one (ABO) analogs containing the piperazine moiety

Prostate cancer (PCa) is a major cause of cancer-related male death in worldwide. To develop of potential anti-prostate cancer agents, 22 kinds of 4-Amino-2H-benzo[h]chromen-2-one analogs were designed and synthesized as potent androgen receptor (AR) antagonist through rational drug modification leading to the discovery of a series of novel antiproliferative compounds. Analogs (3, 4, 5, 7, 8, 10, 11, 12, 16, 18, 21, 23, and 24) exhibited potent antagonistic potency against AR (inhibition >50%), and exhibited potent AR binding affinities as well as displayed the higher activities than finasteride toward LNCaP cells (AR-rich) versus PC-3 cells (AR-deficient). Moreover, the docking study suggested that the most potent antagonist 23 mainly bind to AR ligand binding pocket (LBP) site through Van der Waals’ force interactions. The structure-activity relationship (SAR) of these designed 4-Amino-2H-benzo[h]chromen-2-one analogs was rationally explored and discussed. Collectively, this work provides a potential lead compound for anticancer agent development related to prostate cancer therapy, and took a step forward towards the development of novel and improved AR antagonists.

Synthesis and biological evaluation of arylpiperazine derivatives as potential anti-prostate cancer agents.

A novel scaffold of arylpiperazine derivatives was discovered as potent androgen receptor (AR) antagonist through rational drug designation based on our pre-work, leading to the discovery of a series of new antiproliferative compounds. Compounds 10, 16, 27, 29 and 31 exhibited relatively strong antagonistic potency against AR and exhibited potent AR binding affinities, while compounds 5, 6, 10, 14, 16, 19, 21, 27 and 31 exhibited strong cytotoxic activities against LNCaP cells (AR-rich) as well as also displayed the higher activities than finasteride toward PC-3 (AR-deficient) and DU145 (AR-deficient). Docking study suggested that the most potent antagonist 16 mainly bind to AR ligand binding pocket (LBP) site through hydrogen bonding interactions. The structure-activity relationship (SAR) of these designed arylpiperazine derivatives was rationally explored and discussed. These results indicated that the novel scaffold compounds demonstrated a step towards the development of novel and improved AR antagonists, and promising candidates for future development were identified.

Novel Trifluoromethylated Enobosarm Analogues with Potent Antiandrogenic Activity In Vitro and Tissue Selectivity In Vivo

Prostate cancer often develops antiandrogen resistance, possibly via androgen receptor (AR) mutations, which change antagonists to agonists. Novel therapies with increased anticancer activity, while overcoming current drug resistance are urgently needed. Enobosarm has anabolic effects on muscle and bone while having no effect on the prostate. Here, we describe the activity of novel chemically modified enobosarm analogues. The rational addition of bis-trifluoromethyl groups into ring B of enobosarm, profoundly modified their activity, pharmacokinetic and tissue distribution profiles. These chemical structural modifications resulted in an improved AR binding affinity-by increasing the molecular occupational volume near helix 12 of AR. In vitro, the analogues SK33 and SK51 showed very potent antiandrogenic activity, monitored using LNCaP/AR-Luciferase cells where growth, PSA and luciferase activity were used as AR activity measurements. These compounds were 10-fold more potent than bicalutamide and 100-fold more potent than enobosarm within the LNCaP model. These compounds were also active in LNCaP/BicR cells with acquired bicalutamide resistance. In vivo, using the AR-Luc reporter mice, these drugs showed potent AR inhibitory activity in the prostate and other AR-expressing tissues, e.g., testes, seminal vesicles, and brain. These compounds do not inhibit AR activity in the skeletal muscle, and spleen, thus indicating a selective tissue inhibitory profile. These compounds were also active in vivo in the Pb-Pten deletion model. SK33 and SK51 have significantly different and enhanced activity profiles compared with enobosarm and are ideal candidates for further development for prostate cancer therapy with potentially fewer side effects. Mol Cancer Ther; 17(9); 1846-58. ©2018 AACR.

Theranostic Pt(IV) Conjugate with Target Selectivity for Androgen Receptor.

It is difficult to diagnose and treat castration-resistant prostate cancer (CRPC) which occurs due to the overexpression of androgen receptor (AR). Because there is a high level of AR in CRPC, we designed and prepared three Pt(IV)-based prodrugs targeting AR. Among them, compound 3, a three-in-one hybrid (an AR binding ligand, a cisplatin unit, and a coumarin moiety), was found to display satisfactory AR binding affinity and antagonist activity against androgen receptor, which could also be effectively internalized and visualized in LNCaP (AR+) cells. Due to its AR affinity, 3 selectively accumulated in greater quantities in LNCaP (AR+) cells than in PC-3 (AR-) cells. Moreover, compound 3 exhibited excellent anticancer activity superior to cisplatin.These results highlight the targeting theranostic application of Pt(IV) prodrugs.

Design, synthesis and biological evaluation of novel 3-oxo-4-oxa-5α-androst-17β-amide derivatives as dual 5α-reductase inhibitors and androgen receptor antagonists

Prostate cancer (PCa) is the second leading cause of death in men. Recently, some researches have showed that 5α-reductase inhibitors were beneficial in PCa treatment as well. In this study, a series of novel 3-oxo-4-oxa-5α-androst-17β-amide derivatives have been designed and synthesized in a more simple and convenient method. Most of the synthesized compounds displayed good 5α-reductase inhibitory activities and androgen receptor binding affinities. Their anti-proliferation activities in PC-3 and LNCaP cell lines were also evaluated and the results indicated that most of the synthesized compounds exhibited potent anti-proliferative activities. It is obvious that the androgen-dependent cell line LNCaP was much more sensitive than the androgen-independent cell line PC-3. Among all the synthesized compounds, 11d and 11k displayed the best inhibition activity with 4-fold more sensitive toward LNCaP than PC-3, which was consistent with their high affinities observed in AR binding assay. Molecular modeling studies suggested that 11k could bind to AR in a manner similar to the binding of dihydrotestosterone to AR. Compared to the finasteride, 11k showed a longer plasma half-life (4h) and a better bioavailability. Overall, based on biological activities data, compound 11d and 11k can be identified as potential dual 5α-reductase inhibitors and AR antagonists which might be of therapeutic importance for prostate cancer treatment.

Efficacy and safety of enzalutamide (ENZA) monotherapy in hormone-naive prostate cancer (HNPC).

5001 Background: In locally-advanced prostate cancer, the antiandrogen bicalutamide (Bic) is used to maintain quality of life relative to castration therapy (LHRHa), but efficacy as a monotherapy is limited. ENZA is an oral androgen receptor (AR) inhibitor with higher AR–binding affinity vs Bic, and it prevents nuclear translocation, shows no DNA binding, and induces apoptosis. ENZA was approved in the US after prolonging overall survival in post-docetaxel metastatic castration resistant prostate cancer. This phase 2 study assessed ENZA monotherapy in patients (pts) with HNPC and noncastrate testosterone (T) ≥230 ng/dL. Methods: Pts with any stage HNPC (ECOG PS 0, life expectancy &gt;1 y) requiring hormonal therapy received ENZA 160 mg/d for 25 wks. Primary endpoint was PSA response (≥80% decline at wk 25). Other endpoints were endocrine levels, pharmacokinetics, safety, and metabolic changes (body composition, bone biomarkers, lipids, and glycemic profiles). Results: 67 pts were enrolled. Median age was 73 y; 39% had metastases, 36% and 24% had prior prostatectomy and radiation, respectively. ENZA levels reached steady state after ~4 wks. Mean changes in metabolic outcomes at wk 25 included: –0.24% total body bone mineral density (BMD), –4.15% lean body mass, 6.85% fat body mass, 14.75% bone alkaline phosphatase, 4.55% total cholesterol, 6.48% triglycerides, –1.98% A1c, –0.10% fasting glucose, and 45.06% HOMA-IR. At wk 25, PSA response was 93% (62/67; 95% CI, 86%–99%); median PSA decrease was –99.6%. Mean T and estrogen increased 114% and 72%, respectively; other endocrine increases were observed, the highest was 185% for luteinizing hormone. Most common treatment-emergent AEs were grade 1 and included gynecomastia (36%), fatigue (34%), nipple pain (19%), and hot flush (18%). Five pts had serious AEs (none drug related). Conclusions: ENZA monotherapy achieved a high PSA response rate and marked PSA decline with efficacy similar to castration. In contrast to castration, BMD remained stable and metabolic variables (fat body mass, lipid and glycemic profiles) were not substantially impacted with ENZA monotherapy over the 6 month study period. Endocrine changes and AEs were consistent with potent AR inhibition. Clinical trial information: NCT01302041.

Enzalutamide monotherapy: Phase II study results in patients with hormone-naive prostate cancer.

18 Background: Enzalutamide (ENZA) is an oral androgen receptor inhibitor that has been approved in the US and shown to increase overall survival by 4.8 months over a placebo (HR, 0.63) in patients with metastatic castration resistant prostate cancer (CRPC) previously treated with docetaxel (Scher et al, N Engl J Med 2012;367:1187). Compared with bicalutamide in nonclinical studies, enzalutamide had higher androgen receptor–binding affinity, prevented nuclear translocation, showed no DNA binding, and induced apoptosis (Tran et al, Science 2009;324:787). In contrast to previous phase II and III studies that exclusively enrolled patients with CRPC receiving androgen deprivation therapy (ie, testosterone (T) levels ≤50 ng/dL), this phase II study assessed the efficacy and safety of ENZA monotherapy in patients who had never received hormone therapy; presenting with non-castrate T levels (≥230 ng/dL). Methods: This was a 25-wk, open-label, single-arm study of patients with hormone-naïve, histologically confirmed prostate cancer (all stages) requiring hormonal treatment, an ECOG PS score of 0, and a life expectancy &gt;1 y. All patients received ENZA 160 mg/d without concomitment castration. Primary endpoint was PSA response (&gt;80% decrease at wk 25). Secondary endpoints included changes in endocrine levels and safety/tolerability. Results: Among 67 men enrolled, the median (range) age was 73 (48, 86) y; 39% had metastases; 36% and 24% had undergone prostatectomy or radiotherapy before study entry. The PSA response rate (&gt;80% PSA decline at wk 25) was 93%, with a median (range) decrease of −99% (−100, −57) at wk 25. Serum T and estrogen levels increased by a median (range) of 113% (−32, 300) and 58% (−49, 321) at wk 25, respectively, compared with baseline. 82% of men reported drug-related AEs (mostly Grade 1 or 2). Most frequent treatment-emergent AEs included gynaecomastia (36%), fatigue (34%), and hot flush (18%). 7% of men experienced SAEs; none were drug-related. Conclusions: ENZA monotherapy (160 mg) was associated with significant PSA response in nearly all men with hormone-naïve prostate cancer. Endocrine level changes and most common AEs (gynecomastica, fatigue and hot flush) were consistent with potent AR inhibition. Clinical trial information: NCT01302041.

ODM-201, a new generation androgen receptor inhibitor for castration-resistant prostate cancer: Preclinical and phase I data.

65^ Background: Castration resistant prostate cancer (CRPC) is characterized by persistent, high level androgen receptor (AR) expression and resistance to conventional AR inhibitors. ODM-201 is a novel AR inhibitor with unique pharmacologic properties that has shown promising results in preclinical and clinical studies. Methods: AR binding affinity of ODM-201 to wild type AR was determined in cytosolic lysates obtained from ventral prostates of castrated rats using a competition binding assay. Additionally, effects of ODM-201 on the growth of castration resistant VCaP tumors was evaluated. Tumors were established by subcutaneous injection of VCaP prostate cancer cells into male nude mice. After initial tumor growth, mice were castrated. ODM-201 (50 mg/kg QD or BID orally) was initiated upon tumor regrowth. Since the risk of seizures has been reported for some second generation AR inhibitors, ODM-201 concentrations in mouse brain homogenates were studied after repeated oral administration to assess the risk. The clinical effects of ODM-201 (100, 200, 300, 500, 700, 900 mg BID) was examined in a Phase I/II dose-escalation trial in patients (N=24) with progressive metastatic CRPC. Results: ODM-201 binds to wild type AR with superior affinity compared to enzalutamide (Ki of 9 and 39 nM, respectively). In the VCaP CRPC model, ODM-201 significantly (p&lt;0.001 compared to castrated control), and more efficiently than enzalutamide, inhibited tumor growth. After oral administration, tissue/plasma ratio of ODM-201 in mouse brain homogenates was negligible in all studied doses. In the Phase I/II clinical study, ODM-201 (100 to 700 mg results available) was well tolerated, and most commonly reported adverse events were asthenia, diarrhea and nausea. A PSA response (defined as ≥ 50% decrease) was obtained in 13/15 patients evaluable at 12 weeks with all patients achieving partial response or stable disease by RECIST/PCWG2 criteria. Conclusions: ODM-201 is a new generation AR inhibitor with superior preclinical efficacy compared to enzalutamide and bicalutamide, it does not enter the brain in preclinical studies, and it shows very promising activity and no significant toxicity in patients with CRPC. Clinical trial information: NCT01317641.

Design, synthesis, and biological evaluation of 3-aryl-3-hydroxy-1-phenylpyrrolidine derivatives as novel androgen receptor antagonists

We designed and synthesized a series of 3-aryl-3-hydroxy-1-phenylpyrrolidine derivatives D and evaluated their potential as novel androgen receptor (AR) antagonists therapeutically effective against castration-resistant prostate cancer (CRPC). Introduction of a methyl group at the 2-position (R2) of the pyrrolidine ring increased the AR binding affinity. The (2S,3R) configuration of the pyrrolidine ring was favorable for the AR antagonistic activity. It was found that introduction of an amide substituent (R1) and a pyridin-3-yl group (Q) was effective for reducing the AR agonistic activity which appeared during the optimization of lead compound 6. Compound 54 showed potent antitumor effects against a CRPC model of LNCaP-hr cell line in a mouse xenograft, in which bicalutamide exhibited only partial suppression of tumor growth. Thus, the pyrrolidine derivatives such as 54 are novel AR antagonists, and their properties having efficacy against CRPC are distinct from those of a representative first-generation antagonist, bicalutamide.

Characterization of In Vitro Synthesized Equine Metabolites of the Selective Androgen Receptor Modulators S24 and S4

Several selective androgen receptor modulators (SARMs) have been synthesized and investigated in humans, rats, and dogs in the past, but no data are yet available concerning the metabolism of SARMs in horses. The aryl-propionamide-derived drug candidates S24 and S4 (andarine) have a strong androgen receptor binding affinity and show distinctive specific cell answers. Although no SARM drug candidate (aiming for testosterone replacement therapy) has completed clinical trials yet, S4 has been illicitly available via the Internet. These facts led to the prohibition of SARMs by the German equestrian federation, and the (mis)use of such compounds would further represent a doping rule violation in horse racing. In this study, the drug candidates S24 and S4 were subjected to in vitro metabolism experiments with equine liver microsomal preparations from a female Quarter Horse to obtain information about potential target analytes in equine doping control analysis. The enzymatically synthesized metabolites were characterized by liquid chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry. All observed S24 and S4 equine metabolites are in agreement with earlier in vitro and in vivo studies in humans and dogs. Nevertheless, the relative percentage of generated equine metabolites (as determined from the analytes’ response in full-scan chromatography–tandem mass spectrometry and –high-resolution/high-accuracy mass spectrometry measurements) differs considerably from the reported profiles. Although the S24 metabolite pattern is comparably balanced concerning glucuronidated and sulfonated conjugates, the major S4 metabolite was found to be the unconjugated dephenylated compound, with a proportion of more than 90%.

Abstract 2170: Dissecting the oncogenic function of a novel androgen receptor-dependent direct target, cell cycle-related kinase (CCRK), in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related deaths worldwide with a gender prevalence observed in men. Recent genetic studies using knockout mouse models have revealed the causal role of androgen receptor (AR) in hepatocarcinogenesis but the underlying molecular mechanism remains unclear. Here we used genome-wide location and functional analyses to show that cell cycle-related kinase (CCRK) is a direct critical mediator of AR signaling. Chromatin immunoprecipitation microarray identified over 200 high-confidence AR direct target genes in HCC cells of which cell cycle regulators were significantly enriched (n = 21; p &amp;lt; 0.0001). Because CCRK has the highest AR binding affinity amongst the identified cell cycle regulators, its regulation, function and expression in HCC were further investigated. Ligand-activated AR was recruited to the CCRK promoter and increased CCRK expression. AR-induced cell cycle progression was abrogated by siRNA-mediated knockdown of CCRK. On the contrary, over-expression of CCRK rescued the G1 arrest induced by AR knockdown. Ectopic CCRK expression in human immortal liver cells induced anchorage-dependent and -independent growth and tumor formation in immunodeficient mice, whereas CCRK inhibition decreased HCC cell growth in vitro and in vivo; demonstrating the strong oncogenic capacity of CCRK in HCC. Mechanistically, CCRK activated β-catenin/T-cell factor (TCF) signaling through phosphorylation of glycogen synthase kinase-3β to increase the expression of downstream pro-proliferative genes, cyclin D1 and epidermal growth factor receptor. Inhibition of β-catenin/TCF signaling significantly attenuated CCRK-induced cell cycle progression, colony formation and tumorigenicity. Conversely, HCC cell growth inhibition by CCRK knockdown was rescued by constitutively active form of β-catenin or TCF. Importantly, AR, CCRK, and active β-catenin were markedly over-expressed and positively correlated among each other in HCC specimens (p &amp;lt; 0.001). Furthermore, CCRK over-expression was significantly associated with tumor staging and poor disease-free survival of patients (p &amp;lt; 0.05), emphasizing the clinical importance of CCRK in HCC. In conclusion, our findings reveal a novel interplay between AR and β-catenin where activated AR transcriptionally up-regulates CCRK expression, thereby activating β-catenin/TCF signaling to induce aberrant cell proliferation. Dissection of this highly activated AR-CCRK-β-catenin/TCF axis sheds new mechanistic insight into hepatocarcinogenesis and provides novel therapeutic targets for the treatment of this and other male-predominant cancers. Acknowledgements: This study was partially supported by the Research Grant Council General Research Fund (462710) and the Direct Grant from the Chinese University of Hong Kong. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2170. doi:10.1158/1538-7445.AM2011-2170