Structural Development of Androgen Receptor Antagonists Using Phenylferrocene Framework as a Hydrophobic Pharmacophore.

We previously identified nitrophenylferrocenes and cyanophenylferrocenes as promising lead structures of novel androgen receptor (AR) antagonists, based on the structural similarity between ferrocene and the steroidal skeleton. In the present research, we explored the structure-activity relationship (SAR) of phenylferrocene derivatives. Introduction of a hydrophobic substituent such as a chlorine atom at the 2-position or 3-position of phenylferrocene derivatives significantly increased the antagonistic activity toward wild-type AR, and among the synthesized compounds, 3-chloro-4-cyanophenylferrocene (29) exhibited the most potent anti-proliferative activity toward the androgen-dependent growth of SC-3 cells expressing wild-type AR (IC50 14 nM). Like conventional antiandrogens such as hydroxyflutamide, the major active metabolite of flutamide, compound 29 exhibited agonistic activity toward T877A-AR, a mutant AR expressed in human prostate cancer cell line LNCaP. Notably, however, the 2-chloro isomer 27 showed potent antagonistic activity toward wild-type AR (IC50 49 nM) and also exhibited antagonistic activity toward T877A-AR. Our SAR data should prove helpful for the development of new-generation AR antagonists based on phenylferrocene as candidate agents to treat drug-resistant prostate cancer.

An integrated in vitro/in silico approach to assess the anti-androgenic potency of isobavachin.

Isobavachin is a dietary flavanone with multiple biological activities. Our previous research has confirmed the estrogenicity of isobavachin, and this work aims to assess the anti-androgenic potency of isobavachin by an integrated in vitro and in silico approach. Isobavachin can limit the proliferation of prostate cancer cells by inducing a distinct G1 cell-cycle arrest. In addition, isobavachin also significantly represses the transcription of androgen receptor (AR)-downstream targets such as prostate specific antigen. Mechanistically, we demonstrated that isobavachin can disrupt the nuclear translocation of AR and promote its proteasomal degradation. The results of computer simulations showed that isobavachin can stably bind to AR, and the amino acid residue Gln711 may play a critical role in AR binding of both AR agonists and antagonists. In conclusion, this work has identified isobavachin as a novel AR antagonist.

Dual-function antiandrogen/HDACi hybrids based on enzalutamide and entinostat.

The combination of androgen receptor antagonists with histone deacetylase inhibitors (HDACi) has been shown to be more effective than antiandrogens alone in halting growth of prostate cancer cell lines. Here we have designed, synthesized and assessed a series of antiandrogen/HDACi hybrids by combining structural features of enzalutamide with either SAHA or entinostat. The hybrids are demonstrated to maintain bifunctionality using a fluorometric HDAC assay and a bioluminescence resonance energy transfer (BRET) antiandrogen assay. Antiproliferative assays showed that hybrids bearing o-aminoanilide-based HDACi motifs outperformed hydroxamic acid based HDACi's. The hybrids demonstrated selectivity for epithelial cell lines vs. stromal cell lines, suggesting a potentially useful therapeutic window.

The anti-androgenic effect of quercetin on hyperandrogenism and ovarian dysfunction induced in a dehydroepiandrosterone rat model of polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is a multi-factorial endocrine disorder associated with hyperandrogenism. Dehydroepiandrosterone (DHEA) administration to prepubertal rats stimulates androgen biosynthesis and generation of the PCOS model. The present study aimed to evaluate the anti-androgenic effects of quercetin (Q) in comparison with metformin (MET) on hyperandrogenism and ovarian dysfunction in a DHEA-induced PCOS rat model. After induction of PCOS, female rats were allocated into six groups with 7 rats in each group: normal control; PCOS (DHEA), MET (25 mg/kg, oral administration), Q (25 mg/kg, oral administration), DHEA + MET (25 mg/kg, oral administration), and DHEA + Q (25 mg/kg, oral administration) for 28 days. MET and Q individually reduced body weight, serum free testosterone (T) and luteinizing hormone (LH), and LH/follicle-stimulating hormone (FSH) ratio in the PCOS rats. Both treatments elevated estradiol (E2) level, ovarian aromatase protein content, and E2/free T ratio in the PCOS rats. Additionally, MET and Q increased preantral, antral, and preovulatory follicles and corpora lutea counts, while both treatments decreased atretic follicle count and eliminated the formation of cysts in the PCOS rats. MET and Q reduced ovarian Bax and elevated Bcl-2 protein abundance in the PCOS rats. Our study revealed that Q is as effective as MET in reducing hyperandrogenism via decreasing free T level and improving hypothalamic-pituitary-ovarian axis function. The results suggest that MET and Q may enhance E2 concentration, ovarian aromatase protein content, folliculogenesis, and decrease atresia via attenuation of hyperandrogenism in PCOS rats.

Development of 2-(5,6,7-Trifluoro-1H-Indol-3-yl)-quinoline-5-carboxamide as a Potent, Selective, and Orally Available Inhibitor of Human Androgen Receptor Targeting Its Binding Function-3 for the Treatment of Castration-Resistant Prostate Cancer.

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.

Polymorphic forms of antiandrogenic drug nilutamide: structural and thermodynamic aspects.

Attempts to obtain new cocrystals of nonsteroidal antiandrogenic drug nilutamide produced alternative polymorphic forms of the compound (Form II and Form III) and their crystal structures were elucidated by single-crystal X-ray diffraction. Apart from the cocrystallization technique, lyophilization was found to be an effective strategy for achieving polymorph control of nilutamide, which was difficult to obtain by other methods. The physicochemical properties and relative stability of the commercial Form I and newly obtained Form II were comprehensively investigated by a variety of analytical methods (thermal analysis, solution calorimetry, solubility, and sublimation), whereas for Form III, only a handful of experimental parameters were obtained due to the elusive nature of the polymorph. Form I and Form II were found to be monotropically related, with Form I being confirmed as the thermodynamically most stable solid phase. In addition, the performance of different DFT-D and semi-empirical schemes for lattice energy calculation and polymorph energy ranking was compared and analysed. Lattice energy calculations using periodic DFT at B3LYP-D3/6-31(F+)G(d,p) and PBEh-3c/def2-mSVP levels of theory were found to provide the most accurate lattice energy values for Form I against experimental data, while PIXEL and PBEh-3c/def2-mSVP were the only methods that predicted the correct order of stability of Forms I and II.

Chemical Degradation of Androgen Receptor (AR) Using Bicalutamide Analog-Thalidomide PROTACs.

A series of PROTACs (PROteolysis-TArgeting Chimeras) consisting of bicalutamide analogs and thalidomides were designed, synthesized, and biologically evaluated as novel androgen receptor (AR) degraders. In particular, we found that PROTAC compound 13b could successfully demonstrate a targeted degradation of AR in AR-positive cancer cells and might be a useful chemical probe for the investigation of AR-dependent cancer cells, as well as a potential therapeutic candidate for prostate cancers.

Improving Strategies in the Development of Protein-Downregulation-Based Antiandrogens.

The androgen receptor (AR) plays a crucial role in the occurrence and development of prostate cancer (PCa), and its signaling pathway remains active in castration-resistant prostate cancer (CRPC) patients. The resistance against antiandrogen drugs in current clinical use is a major challenge for the treatment of PCa, and thus the development of new generations of antiandrogens is under high demand. Recently, strategies for downregulating the AR have attracted significant attention, given its potential in the discovery and development of new antiandrogens, including G-quadruplex stabilizers, ROR-γ inhibitors, AR-targeting proteolysis targeting chimeras (PROTACs), and other selective AR degraders (SARDs), which are able to overcome current resistance mechanisms such as acquired AR mutations, the expression of AR variable splices, or overexpression of AR. This review summarizes the various strategies for downregulating the AR protein, at either the mRNA or protein level, thus providing new ideas for the development of promising antiandrogen drugs.

Structure of human steroid 5α-reductase 2 with the anti-androgen drug finasteride.

Human steroid 5α-reductase 2 (SRD5A2) is an integral membrane enzyme in steroid metabolism and catalyzes the reduction of testosterone to dihydrotestosterone. Mutations in the SRD5A2 gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride, as SRD5A2 inhibitors, are widely used antiandrogen drugs for benign prostate hyperplasia. The molecular mechanisms underlying enzyme catalysis and inhibition for SRD5A2 and other eukaryotic integral membrane steroid reductases remain elusive due to a lack of structural information. Here, we report a crystal structure of human SRD5A2 at 2.8 Å, revealing a unique 7-TM structural topology and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride in a largely enclosed binding cavity inside the transmembrane domain. Structural analysis together with computational and mutagenesis studies reveal the molecular mechanisms of the catalyzed reaction and of finasteride inhibition involving residues E57 and Y91. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region that regulate NADPH/NADP+ exchange. Mapping disease-causing mutations of SRD5A2 to our structure suggests molecular mechanisms for their pathological effects. Our results offer critical structural insights into the function of integral membrane steroid reductases and may facilitate drug development.

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.

Complete NMR assignment and absolute configuration of k4610422, a norditerpenoid inhibitor of testosterone-5α-reductase originally from Streptosporangium: rediscovery from a thermophilic Actinomadura.

A norditerpenoid k4610422 (1), an inhibitor of testosterone-5α-reductase originally discovered from a mesophilic rare actinomycete of the genus Streptosporangium, was isolated from the culture extract of a thermophilic actinomycete Actinomadura sp. The complete 1H and 13C NMR assignment and absolute configuration of 1 were addressed by spectroscopic measurements including NOESY and CD spectra coupled with ECD calculation, which allowed to establish the (5 R,9 S,10 R,13 S)-configuration. Compound 1 was moderately cytotoxic against P388 murine leukemia cells with IC50 30 µM.

Gonadal Hormones and Bone.

In both sexes, estrogen is one of the most essential hormones for maintaining bone integrity. Also, especially in men, androgen has beneficial effects on bone independent of estrogen. However, estrogen replacement therapy for postmenopausal women increases the risk of developing breast cancer and endometrial cancer, and androgen replacement therapy for partial androgen deficiency of the aging male increases the risk of developing prostate cancer. Various mechanisms have been proposed on the effects of gonadal hormones on bone, such as effects through cytokines including IL-6 and effects on the OPG/RANKL ratio. In addition, large amounts of new information deriving from high-throughput gene expression analysis raise the possibility of multiple other effects on bone cells. Both estrogen and androgen exert their effects via the estrogen receptor (ER) or the androgen receptor (AR), which belongs to the nuclear receptor superfamily. Compounds such as selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) also bind ER and AR, respectively. However, SERMs and SARMs alter the ER or AR structure differently from estrogen or androgen, resulting in other downstream gene responses. As a result they can exert favorable effects on bone while suppressing the undesirable actions of estrogen and androgen. Elucidation of ER and AR ligand-specific and tissue-specific gene regulation mechanisms will also provide information on the signal transduction mechanisms of other nuclear receptors and will be valuable for the development of new therapeutic agents.

A Structure-Activity Relationship Study of Bis-Benzamides as Inhibitors of Androgen Receptor-Coactivator Interaction.

The interaction between androgen receptor (AR) and coactivator proteins plays a critical role in AR-mediated prostate cancer (PCa) cell growth, thus its inhibition is emerging as a promising strategy for PCa treatment. To develop potent inhibitors of the AR-coactivator interaction, we have designed and synthesized a series of bis-benzamides by modifying functional groups at the N/C-terminus and side chains. A structure-activity relationship study showed that the nitro group at the N-terminus of the bis-benzamide is essential for its biological activity while the C-terminus can have either a methyl ester or a primary carboxamide. Surveying the side chains with various alkyl groups led to the identification of a potent compound 14d that exhibited antiproliferative activity (IC50 value of 16 nM) on PCa cells. In addition, biochemical studies showed that 14d exerts its anticancer activity by inhibiting the AR-PELP1 interaction and AR transactivation.

Design and synthesis of novel steroidal imidazoles as dual inhibitors of AR/CYP17 for the treatment of prostate cancer.

Both AR and CYP17 are important targets for blocking androgen signaling, and it has been accepted that multifunctional drugs have a low risk of drug resistance in the treatment of cancer. Thus, herein a series of steroidal imidazoles were designed, synthesized and evaluated as dual AR/CYP17 ligands. Several compounds displayed good biological profiles in both enzymatic and cellular assays. SAR studies showed that introducing oximino at the C-3 position of steroidal scaffold is beneficial to the enhancement of AR antagonistic activity. Among these compounds, the most potent compound 13a exhibited the best AR inhibition (IC50 = 0.5 µM) that was 27-fold increase compared with the hit compound 5 as well as comparable CYP17 inhibition (IC50 = 11 µM). Additionally, 13a displayed promising anti-proliferative effects on LNCap cell lines with the IC50 value of 23 µM which was superior to positive control Flutamide (IC50 = 28 µM). Furthermore, the docking results of 13a revealed that the oxygen atom at the position of C-3 connected to the heme of CYP17, which may be helpful for its satisfactory dual-target inhibition. In summary, this study provides an efficient strategy for multi-targeting drug discovery in the treatment of prostate cancer.

Broadband dielectric spectroscopy as an experimental alternative to calorimetric determination of the solubility of drugs into polymer matrix: Case of flutamide and various polymeric matrixes.

In this paper we determined the solubility limits of the amorphous flutamide within the two different polymeric matrixes - poly vinylpyrrolidone and poly vinylacetate. In order to achieve this goal, series of broadband dielectric spectroscopy measurements were performed. As a result we found that the maximal amount of the drug that can be successfully dissolved within the PVAc (maintaining the non-supersaturated conditions) is equal to 35 wt% of the amorphous solid dispersion system. Interestingly enough similar results, in terms of solubility limits, were achieved utilizing significantly higher amount of the pharmaceutical - 71 wt% - in the PVP matrix. Accordingly, we established the following relationship in the solubility limits of the amorphous flutamide dispersed within examined polymer matrixes: PVP > PVAc. It is worth highlighting that in order to preserve the thermodynamic stability - one of the two contributors to the physical stability - drug loading in the amorphous solid dispersion system should not exceed its solubility limits. Hence, choosing appropriate amount of the polymer addition will determine if obtained system remains physically stable. Subsequently, we presented the "stability maps" for all investigated FL-based ASD systems from which one might predict the stabilization effect exerted by certain amount of polymer.

TiO2 Photocatalyzed Oxidation of Drugs Studied by Laser Ablation Electrospray Ionization Mass Spectrometry.

In drug discovery, it is important to identify phase I metabolic modifications as early as possible to screen for inactivation of drugs and/or activation of prodrugs. As the major class of reactions in phase I metabolism is oxidation reactions, oxidation of drugs with TiO2 photocatalysis can be used as a simple non-biological method to initially eliminate (pro)drug candidates with an undesired phase I oxidation metabolism. Analysis of reaction products is commonly achieved with mass spectrometry coupled to chromatography. However, sample throughput can be substantially increased by eliminating pretreatment steps and exploiting the potential of ambient ionization mass spectrometry (MS). Furthermore, online monitoring of reactions in a time-resolved way would identify sequential modification steps. Here, we introduce a novel (time-resolved) TiO2-photocatalysis laser ablation electrospray ionization (LAESI) MS method for the analysis of drug candidates. This method was proven to be compatible with both TiO2-coated glass slides as well as solutions containing suspended TiO2 nanoparticles, and the results were in excellent agreement with studies on biological oxidation of verapamil, buspirone, testosterone, andarine, and ostarine. Finally, a time-resolved LAESI MS setup was developed and initial results for verapamil showed excellent analytical stability for online photocatalyzed oxidation reactions within the set-up up to at least 1 h. Graphical Abstract.

Small molecule-induced degradation of the full length and V7 truncated variant forms of human androgen receptor.

The androgen receptor (AR) is a hormone-activated transcription factor that regulates the development and progression of prostate cancer (PCa) and represents one of the most well-established drug targets. Currently clinically approved small molecule inhibitors of AR, such as enzalutamide, are built upon a common chemical scaffold that interacts with the AR by the same mechanism of action. These inhibitors eventually fail due to the emergence of drug-resistance in the form of AR mutations and expression of truncated AR splice variants (e.g. AR-V7) that are constitutively active, signalling the progression of the castration-resistant state of the disease. The urgent need therefore continues for novel classes of AR inhibitors that can overcome drug resistance, especially since AR signalling remains important even in late-stage advanced PCa. Previously, we identified a collection of 10-benzylidene-10H-anthracen-9-ones that effectively inhibit AR transcriptional activity, induce AR degradation and display some ability to block recruitment of hormones to the receptor. In the current work, we extended the analysis of the lead compounds, and used methods of both ligand- and structure-based drug design to develop a panel of novel 10-benzylidene-10H-anthracen-9-one derivatives capable of suppressing transcriptional activity and protein expression levels of both full length- and AR-V7 truncated forms of human androgen receptor. Importantly, the developed compounds efficiently inhibited the growth of AR-V7 dependent prostate cancer cell-lines which are completely resistant to all current anti-androgens.

Therapeutic Potential of Leelamine, a Novel Inhibitor of Androgen Receptor and Castration-Resistant Prostate Cancer.

Clinical management of castration-resistant prostate cancer (CRPC) resulting from androgen deprivation therapy remains challenging. CRPC is driven by aberrant activation of androgen receptor (AR) through mechanisms ranging from its amplification, mutation, post-translational modification, and expression of splice variants (e.g., AR-V7). Herein, we present experimental evidence for therapeutic vulnerability of CRPC to a novel phytochemical, leelamine (LLM), derived from pine tree bark. Exposure of human prostate cancer cell lines LNCaP (an androgen-responsive cell line with mutant AR), C4-2B (an androgen-insensitive variant of LNCaP), and 22Rv1 (a CRPC cell line with expression of AR-Vs), and a murine prostate cancer cell line Myc-CaP to plasma achievable concentrations of LLM resulted in ligand-dependent (LNCaP) and ligand-independent (22Rv1) growth inhibition in vitro that was accompanied by downregulation of mRNA and/or protein levels of full-length AR as well as its splice variants, including AR-V7. LLM treatment resulted in apoptosis induction in the absence and presence of R1881. In silico modeling followed by luciferase reporter assay revealed a critical role for noncovalent interaction of LLM with Y739 in AR activity inhibition. Substitution of the amine group with an isothiocyanate functional moiety abolished AR and cell viability inhibition by LLM. Administration of LLM resulted in 22Rv1 xenograft growth suppression that was statistically insignificant but was associated with a significant decrease in Ki-67 expression, mitotic activity, expression of full-length AR and AR-V7 proteins, and secretion of PSA. This study identifies a novel chemical scaffold for the treatment of CRPC. Mol Cancer Ther; 17(10); 2079-90. ©2018 AACR.

Efficient Lead Finding, Activity Enhancement and Preliminary Selectivity Control of Nuclear Receptor Ligands Bearing a Phenanthridinone Skeleton.

BACKGROUND: Nuclear receptors (NRs) are considered as potential drug targets because they control diverse biological functions. However, steroidal ligands for NRs have the potential to cross-react with other nuclear receptors, so development of non-steroidal NR ligands is desirable to obtain safer agents for clinical use. We anticipated that efficient lead finding and enhancement of activity toward nuclear receptors recognizing endogenous steroidal ligands might be achieved by exhaustive evaluation of a steroid surrogate library coupled with examination of structure-activity relationships (SAR). METHOD: We evaluated our library of RORs (retinoic acid receptor-related orphan receptors) inverse agonists and/or PR (progesterone receptor) antagonists based on the phenanthridinone skeleton for antagonistic activities toward liver X receptors (LXRs), androgen receptor (AR) and glucocorticoid receptor (GR) and examined their SAR. RESULTS: Potent LXRß, AR, and GR antagonists were identified. SAR studies led to a potent AR antagonist (IC50: 0.059 µM). CONCLUSIONS: Our approach proved effective for efficient lead finding, activity enhancement and preliminary control of selectivity over other receptors. The phenanthridinone skeleton appears to be a promising steroid surrogate.

Do progestins contribute to (anti-)androgenic activities in aquatic environments?

Unknown compounds with (anti-)androgenic activities enter the aquatic environment via municipal wastewater treatment plants (WWTPs). Progestins are well-known environmental contaminants capable of interfering with androgen receptor (AR) signaling pathway. The aim of the present study was to determine if 15 selected progestins have potential to contribute to (anti-)androgenic activities in municipal wastewaters and the respective recipient surface waters. AR-specific Chemically Activated LUciferase gene eXpression bioassay in agonistic (AR-CALUX) and antagonistic (anti-AR-CALUX) modes and liquid chromatography tandem atmospheric pressure chemical ionization/atmospheric photoionization with hybrid quadrupole/orbital trap mass spectrometry operated in high resolution product scan mode (LC-APCI/APPI-HRPS) methods were used to assess (anti-)androgenic activity and to detect the target compounds, respectively. The contribution of progestins to (anti-)androgenic activities was evaluated by means of a biologically and chemically derived toxicity equivalent approach. Androgenic (0.08-59 ng/L dihydrotestosterone equivalents - DHT EQs) and anti-androgenic (2.4-26 µg/L flutamide equivalents - FLU EQs) activities and progestins (0.19-75 ng/L) were detected in selected aquatic environments. Progestins displayed androgenic potencies (0.01-0.22 fold of dihydrotestosterone) and strong anti-androgenic potencies (9-62 fold of flutamide). Although they accounted to some extent for androgenic (0.3-29%) and anti-androgenic (4.6-27%) activities in influents, the progestins' contribution to (anti-)androgenic activities was negligible (≤2.1%) in effluents and surface waters. We also tested joint effect of equimolar mixtures of target compounds and the results indicate that compounds interact in an additive manner. Even if progestins possess relatively strong (anti-)androgenic activities, when considering their low concentrations (sub-ng/L to ng/L) it seems unlikely that they would be the drivers of (anti-)androgenic effects in Czech aquatic environments.