Synthesis and preliminary investigations into novel 1,2,3-triazole-derived androgen receptor antagonists inspired by bicalutamide

A versatile and high yielding synthesis of novel androgen receptor (AR) antagonists is presented. Using this methodology, six 1,4-substituted-1,2,3-triazole derived bicalutamide mimics were synthesised in five steps and in isolated overall yields from 41% to 85%. Evaluation of these compounds for their anti-proliferative properties against androgen dependent (LNCaP) and independent (PC-3) cells showed promising IC₅₀ values of 34–45 μM and 29–151 μM, respectively. The data suggest that the latter compounds may be an excellent starting point for the development of prostate cancer therapeutics for both androgen dependent and independent forms of this disease. Docking of these compounds (each enantiomer) in silico into the T877A mutated androgen receptor, as possessed by LNCaP cells, was also undertaken.     

Small-molecule androgen receptor downregulators as an approach to treatment of advanced prostate cancer

Chemical starting points were investigated for downregulation of the androgen receptor as an approach to treatment of advanced prostate cancer. Although prototypic steroidal downregulators such as 6a designed for intramuscular administration showed insufficient cellular potency, a medicinal chemistry program derived from a novel androgen receptor ligand 8a led to 6-[4-(4-cyanobenzyl)piperazin-1-yl]-3-(trifluoromethyl)[1,2,4]triazolo[4,3-b]pyridazine (10b), for which high plasma levels following oral administration in a preclinical model compensate for moderate cellular potency.     

DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation

The majority of human prostate cancer cell lines, including the two "classical" cell lines DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. However, other studies have provided evidence that the DU-145 and PC-3 cell lines express AR mRNA. These contradictory observations prompted us to investigate whether DU-145 and PC-3 cell lines express the androgen receptor. Using antipeptide antibodies directed against three distinct regions of the human AR protein and an improved method to detect AR protein in immunoblotting, we report that DU-145 and PC-3 cell lines express AR protein. We found that the relative levels of the AR mRNA and protein that were detected in DU-145 and PC-3 cell lines were lower than the LNCaP, an AR-positive cell line. Moreover, the antibody directed against the non-variant region (amino acids 299-315), but not the variant N- or C-terminal region (amino acids 1-20 and 900-919, respectively) of the human AR protein, detected the expression of AR in all prostate cancer cell lines. Notably, treatment of these cell lines with dihydrotestosterone (DHT) resulted in measurable increases in the AR protein levels and considerable nuclear accumulation. Although, treatment of DU-145 and PC-3 cells with DHT did not result in stimulation of the activity of an AR-responsive reporter, knockdown of AR expression in PC-3 cells resulted in decreases in p21(CIP1) protein levels, and a measurable decrease in the activity of the p21-luc-reporter. Our observations demonstrate the expression of AR protein in DU-145 and PC-3 prostate cancer cell lines.     

The androgen receptor: a potential target for therapy of prostate cancer

The androgen receptor plays a pivotal role in the prostate. Its primary function is to provide responsive gene products for differentiation and growth, but under abnormal conditions it contributes to the development of prostate cancer. The goal of this review is to elucidate the molecular functions of the androgen receptor and its role in prostate cancer. Initially the function of the androgen receptor will be described. Next, the clinical diagnosis, epidemiological impact, and treatments of androgen-dependent and -independent prostate cancer will be discussed. Finally we will examine how the mechanism of androgen action has played a role in the translation of new therapies and how this may influence future treatment modalities of prostate cancer.     

Microbubble-enhanced ultrasound to deliver an antisense oligodeoxynucleotide targeting the human androgen receptor into prostate tumours

We have shown recently that downregulation of the androgen receptor (AR), one of the key players in prostate tumor cells, with short antisense oligodeoxynucleotides (ODNs) results in inhibition of prostate tumor growth. Particularly with regard to an application of these antisense drugs in vivo, we now investigated the usefulness of microbubble-enhanced ultrasound to deliver these ODNs into prostate cancer cells.

Our short antisense AR ODNs were loaded onto the lipid surface of cationic gas-filled microbubbles by ion charge binding, and delivered into the cells by bursting the loaded microbubbles with ultrasound. In vitro experiments were initially performed to show that this kind of delivery system works in principle. In fact, transfection of prostate tumor cells with antisense AR ODNs using microbubble-enhanced ultrasound resulted in 49% transfected cells, associated with a decrease in AR expression compared to untreated controls. In vivo, uptake of a digoxigenin-labelled ODN was found in prostate tumour xenografts in nude mice following intratumoral or intravenous injection of loaded microbubbles and subsequent exposure of the tumour to ultrasound, respectively. Our results show that ultrasound seems to be the driving force of this delivery system. Uptake of the ODN was also observed in tumors after treatment with ultrasound alone, with only minor differences compared to the combined use of microbubbles and ultrasound.     

The synthesis and evaluation of [2.2.1]-bicycloazahydantoins as androgen receptor antagonists

A novel series of [2.2.1]-azahydantoins has been designed and synthesized in an enantiospecific manner. The ability of these compounds to act as antagonists to the androgen receptor was investigated and several were found to have potent activity in vitro.     

Pomegranate polyphenols down-regulate expression of androgen-synthesizing genes in human prostate cancer cells overexpressing the androgen receptor

Prostate cancer is dependent on circulating testosterone in its early stages and is treatable with radiation and surgery. However, recurrent prostate tumors advance to an androgen-independent state in which they progress in the absence of circulating testosterone, leading to metastasis and death. During the development of androgen independence, prostate cancer cells are known to increase intracellular testosterone synthesis, which maintains cancer cell growth in the absence of significant amounts of circulating testosterone. Overexpression of the androgen receptor (AR) occurs in androgen-independent prostate cancer and has been proposed as another mechanism promoting the development of androgen independence. The LNCaP-AR cell line is engineered to overexpress AR but is otherwise similar to the widely studied LNCaP cell line. We have previously shown that pomegranate extracts inhibit both androgen-dependent and androgen-independent prostate cancer cell growth. In this study, we examined the effects of pomegranate polyphenols, ellagitannin-rich extract and whole juice extract on the expression of genes for key androgen-synthesizing enzymes and the AR. We measured expression of the HSD3B2 (3beta-hydroxysteroid dehydrogenase type 2), AKR1C3 (aldo-keto reductase family 1 member C3) and SRD5A1 (steroid 5alpha reductase type 1) genes for the respective androgen-synthesizing enzymes in LNCaP, LNCaP-AR and DU-145 human prostate cancer cells. A twofold suppression of gene expression was considered statistically significant. Pomegranate polyphenols inhibited gene expression and AR most consistently in the LNCaP-AR cell line (P=.05). Therefore, inhibition by pomegranate polyphenols of gene expression involved in androgen-synthesizing enzymes and the AR may be of particular importance in androgen-independent prostate cancer cells and the subset of human prostate cancers where AR is up-regulated.     

The androgen receptor (AR) in syndromes of androgen insensitivity and in prostate cancer

The actions of androgens, principally testosterone and 5alpha-dihydrotestosterone, are mediated by a specific receptor protein, the androgen receptor (AR), which is encoded by a single-copy gene located on the human X-chromosome. This receptor protein is a prototypical member of the nuclear receptor family and modulates a range of processes during embryogenesis and in the adult. During embryogenesis, normal AR function is critical to the development of the male phenotype and defects of the AR cause a range of phenotypic abnormalities of male sexual development. Complete loss of AR function has been traced to a number of distinct types of genetic events, including abnormalities of mRNA splicing, the introduction of premature termination codons, and amino acid substitution mutations. An interesting subset of mutations is that in which the AR is completely undetectable using sensitive immunoassays. In all instances, these functional abnormalities are associated with a phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, partial defects of AR function are almost invariably caused by amino acid substitutions within the DNA- and hormone-binding domains of the receptor protein. Such partial defects of receptor function may be caused by changes in either receptor function or receptor abundance.The alterations of AR function and expression that have been characterized in clinical prostatic cancers and in prostate cancer cell lines differ in several important respects. A number of studies have documented the emergence of considerable heterogeneity of AR expression at early stages in the development of prostate cancer. Despite these early changes of AR expression, a substantial body of information suggests that the AR is expressed in advanced forms of prostate cancer, in some cases as the result of amplification events. While infrequent in localized tumors, mutations of the AR have been identified in a number of advanced prostatic cancers and in some instances appear to alter the ligand specificity of the AR. Finally, it appears that other signaling pathways can act to influence AR function.     

Triterpenes from Alisma orientalis act as androgen receptor agonists,progesterone receptor antagonists,and glucocorticoid receptor antagonists

Alisma orientalis, a well-known traditional medicine, exerts numerous pharmacological effects including anti-diabetes, anti-hepatitis, and anti-diuretics but its bioactivity is not fully clear. Androgen receptor (AR), progesterone receptor (PR), and glucocorticoid receptor (GR) are three members of nuclear receptor superfamily that has been widely targeted for developing treatments for essential diseases including prostate cancer and breast cancer. In this study, two triterpenes, alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis were determined whether they may act as androgen receptor (AR), progesterone receptor (PR), or glucocorticoid receptor (GR) modulators. Indeed, in the transient transfection reporter assays, alisol M 23-acetate and alisol A 23-acetate transactivated AR in dose-dependent manner, while they transrepressed the transactivation effects exerted by agonist-activated PR and GR. Through molecular modeling docking studies, they were shown to respectively interact with AR, PR, or GR ligand binding pocket fairly well. All these results indicate that alisol M 23-acetate and alisol A 23-acetate from Alisma orientalis might possess therapeutic effects through their modulation of AR, PR, and GR pathways.     

GLI1, a crucial mediator of sonic hedgehog signaling in prostate cancer, functions as a negative modulator for androgen receptor

Sonic hedgehog (SHH) signaling, acting in a combinatorial manner with androgen signaling, is essential for prostate patterning and development. Recently, elevated activation of SHH signaling has been shown to play important roles in proliferation, progression and metastasis of prostate cancer. In this report, we demonstrate for the first time, that GLI1, which has been shown to play a central role in SHH signaling in prostate cancer, can act as a co-repressor to substantially block androgen receptor (AR)-mediated transactivation, at least in part, by directly interacting with AR. Our observations suggest that the SHH-GLI pathway might be one of determinants governing the transition of prostate cancer from an androgen-dependent to an androgen-independent state by compensating, or even superseding androgen signaling.     

Structure-activity relationship analysis of 3-phenylpyrazole derivatives as androgen receptor antagonists

Abstract

Prostate cancer (PCa), an epithelial malignancy that occurs in the prostate, reminds the second leading cause of cancer-related incidence in men worldwidely. Androgen receptor antagonists are the main therapeutic strategy of PCa, which can block the binding of androgen to androgen receptors. However, the long-term treatment of marketed anti-androgens in patients can inevitably cause drug resistance problem. The research of searching for new drugs with novel skeleton is always on the way. Recently, a series of 3-phenylpyrazole derivatives were reported to antagonize the function of AR, but their efficiencies are not good enough and need to be improved. In this work, comparative molecular field analysis and comparative molecular similarity indices analysis methods were employed to study the structure activity relationships of these derivatives. Two different methods were used to obtain the optimal molecular conformation alignments, one is based on atomic alignment and the other is based on molecular docking. The final result shows that both these two strategies can obtain satisfactory results and the atomic alignment performs a little better than docking. The models illustrate the key structural features highly related with the androgenic bioactivity and provide valuable suggestions for the design of new androgen receptor antagonists in future.

Communicated by Ramaswamy H. Sarma     

Discovery of novel free fatty acid receptor 1 agonists bearing triazole core via click chemistry

The free fatty acid receptor 1 (FFA1/GPR40) is a novel antidiabetic target based on particular mechanism in enhancing glucose-stimulated insulin secretion. Most of reported FFA1 agonists, however, have been suffered from relatively high lipophilicity and molecular weight. Aiming to develop potent agonists with improved physicochemical property, 25 compounds containing triazole scaffold and various carboxylic acid fragments were synthesized via the click chemistry. Among them, the optimal lead compound 26 with relatively low lipophicity (Log D_7.4 = 1.95) and molecular weight (Mw = 391.78) exhibited a considerable FFA1 agonistic activity (36.15%). In addition, compound 26 revealed a significant improvement in the glucose tolerance with a 21.4% and 14.2% reduction of glucose AUC_0–2h in normal ICR mice and type 2 diabetic C57BL/6 mice, respectively. All of these results demonstrated that compound 26 was considered to be a promising lead compound suitable for further optimization.     

Pantolactams as androgen receptor antagonists for the topical suppression of sebum production

A series of pantolactam based compounds were identified as potent antagonists for the androgen receptor (AR). Those that possessed properties suitable for topical delivery were evaluated in the validated Hamster Ear Model. Several compounds were found to be efficacious in reducing wax esters, a major component of sebum, initiating further preclinical work on these compounds.     

Proteomic analysis of the androgen receptor via MS-compatible purification of biotinylated protein on streptavidin resin

The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding.     

Capsaicin, a component of red peppers, induces expression of androgen receptor via PI3K and MAPK pathways in prostate LNCaP cells

In this study, capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) induced an increase in the cell viability of the androgen-responsive prostate cancer LNCaP cells, which was reversed by the use of the TRPV1 antagonists capsazepine, I-RTX and SB 366791. In further studies we observed that capsaicin induced a decrease in ceramide levels as well as Akt and Erk activation. To investigate the mechanism of capsaicin action we measured androgen (AR) receptor levels. Capsaicin induced an increase in the AR expression that was reverted by the three TRPV1 antagonists. AR silencing by the use of siRNA, as well as blocking the AR receptor with bicalutamide, inhibited the proliferative effect of capsaicin.     

Oxidative stress and androgen receptor signaling in the development and progression of castration-resistant prostate cancer

Aberrant androgen receptor (AR) signaling plays a critical role in androgen-dependent prostate cancer (PCa), as well as in castration-resistant PCa (CRPC). Oxidative stress seems to contribute to the tumorigenesis and progression of PCa, as well as the development of CRPC, via activation of AR signaling. This notion is supported by the fact that there is an aberrant or improper regulation of the redox status in these disorders. Additionally, androgen-deprivation-induced oxidative stress seems to be involved in the pathogenesis of several disorders caused by androgen-deprivation therapy (ADT), including osteoporosis, neurodegenerative disease, and cardiovascular disease. Oxidative stress can be suppressed with antioxidants or via a reduction in reactive oxygen species production. Thus, developing new therapeutic agents that reduce oxidative stress might be useful in preventing the conversion of androgen-dependent PCa into CRPC, as well as reducing the adverse effects associated with ADT. The objective of this review is to provide an overview regarding the relationship between oxidative stress and AR signaling in the context of PCa and especially CRPC. Additionally, we discuss the potential use of antioxidant therapies in the treatment of PCa.