3beta-hydroxy-17-(1h-benzimidazole-1-yl)androsta-5-16-diene and Prostatic-Neoplasms

In this review, we summarize the importance of AR variants with a particular focus on clinically relevant members of this family.. A non-systematic literature review was performed based on Medline and PubMed.. Endocrine therapy represents the central paradigm for the management of prostate cancer. Eventually, in response to androgen ablation therapy, several resistance mechanisms against the endocrine therapy might develop that can circumvent the therapy approaches. One specific resistance mechanism that has gained increasing attention is the generation of alternatively spliced variants of the androgen receptor, with AR-V7 being the most prominent. More broadly, AR-V7 is one member of a group of alternatively spliced AR variants that share a common feature, the missing ligand-binding domain. These ΔLBD androgen receptor variants have shown the capability to induce androgen receptor-mediated gene transcription even under conditions of androgen deprivation and to drive cancer progression.. The methods used for detecting AR-Vs, at least on the mRNA level, are well-advanced and harbor the potential to be introduced into clinical diagnostics. It is important to note, that the testing, especially of AR-V7 has its limitations in predicting treatment response. More promising is the great number of active clinical trials aimed at reducing the AR-Vs, and using this to re-sensitize CRPC towards endocrine treatment might provide additional treatment options for CRPC patients in the future.

Topics: Alternative Splicing; Androgen Antagonists; Androstadienes; Antineoplastic Agents, Hormonal; Benzamides; Benzhydryl Compounds; Benzimidazoles; Benzoquinones; Binding Sites; Chlorohydrins; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; HSP90 Heat-Shock Proteins; Humans; Isoindoles; Isoxazoles; Lactams, Macrocyclic; Male; Niclosamide; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Protein Domains; Protein Isoforms; Proteins; Receptors, Androgen; Resorcinols; RNA, Messenger

Given prostate cancer is driven, in part, by its responsiveness to androgens, treatments historically employ methods for their removal from circulation. Approaches as crude as castration, and more recently blockade of androgen synthesis or receptor binding, are still of limited use long term, since other steroids of adrenal origin or tumor origin can supersede that role as the 'castration resistant' tumor re-emerges. Broader inhibition of steroidogenesis using relatively nonselective P450 inhibitors such as ketoconazole is not an alternative since a general disruption of steroid biosynthesis is neither safe nor effective. The recent emergence of drugs more selectively targeting CYP17 have been more effective, and yet extension of life has been on the scale of months rather than years. It is now becoming clear this shortcoming arises from the adaptive capabilities of many tumors to initiate local steroid synthesis and/or become responsive to novel early pathway adrenal steroids that are synthesized when lyase activity is not selectively blocked, and ACTH rises in the face of declining cortisol feedback. Abiraterone has been described as a lyase selective inhibitor, yet its use still requires co-administration of prednisone to suppress such a rise of ACTH and fall in cortisol. So is creation of a selective lyase inhibitor even possible? Can C19 steroid production be achieved without a prominent decline in cortisol and corresponding rise in ACTH? Decades of scientific study of CYP17 in humans and nonhuman primates, as well as nature's own experiments of gene mutations in humans, reveal 'true' or 'isolated' 17,20 lyase deficiency does quite selectively prevent C19 steroid biosynthesis whereas simple 17 hydroxylase deficiency also suppresses cortisol. We propose these known outcomes of natural mutations should be used to guide analysis of clinical trials and long term outcomes of CYP17 targeted drugs. In this review, we use that framework to re-evaluate the basic and clinical outcomes of many compounds being used or in development for treatment of castration resistant prostate cancer. Specifically, we include the nonselective drug ketoconazole, and then the CYP17 targeted drugs abiraterone, orteronel (TAK-700), galaterone (TOK-001), and seviteronel (VT-464). Using this framework, we can fully discriminate the clinical outcomes for ketoconazole, a drug with broad specificity, yet clinically ineffective, from that of abiraterone, the first CYP17 targeted therapy

Topics: Adrenal Hyperplasia, Congenital; Androstadienes; Androstenes; Antineoplastic Agents; Benzimidazoles; Cytochrome P-450 CYP3A Inhibitors; Drug Therapy, Combination; Humans; Hydrocortisone; Imidazoles; Ketoconazole; Male; Naphthalenes; Prednisone; Prostate; Prostatic Neoplasms; Protective Factors; Steroid 17-alpha-Hydroxylase; Triazoles

Major advances have been achieved recently in the treatment of metastatic castration-resistant prostate cancer, resulting in significant improvements in quality of life and survival with the use of several new agents, including the next-generation androgen receptor (AR)-targeted drugs abiraterone and enzalutamide. However, virtually all patients will eventually progress on these therapies and most will ultimately die of treatment-refractory metastatic disease. Recently, several mechanisms of resistance to AR-directed therapies have been uncovered, including the AR splice variant 7 (AR-V7), which is a ligand-independent constitutionally-active form of the AR that has been associated with poor outcomes to abiraterone and enzalutamide. Galeterone, a potent anti-androgen with three modes of action (CYP17 lyase inhibition, AR antagonism, and AR degradation), is a novel agent under clinical development that could potentially target both full-length AR and aberrant AR, including AR-V7. In this manuscript, we will first discuss the biological mechanisms of action of galeterone and then review the safety and efficacy data from Phase I and II clinical studies of galeterone in patients with metastatic castration-resistant prostate cancer. A Phase III study of galeterone (compared against enzalutamide) in AR-V7-positive patients is currently underway, and represents the first pivotal trial using a biomarker-selection design in this disease.

Topics: Androstadienes; Antineoplastic Agents; Benzimidazoles; Humans; Male; Prostatic Neoplasms; Receptors, Androgen

The review examines recent advances in second-line hormonal therapy for the treatment of castrate-resistant prostate cancer (CRPC).. Recent data highlight the continued importance of androgen signaling in CRPC. These findings have spurred the development of novel inhibitors of adrenal and intra-tumoral androgen synthesis and novel androgen signaling inhibitors with activity in CRPC. In the past year abiraterone acetate, a CYP17 (17α-hydroxylase/17, 20 lyase) inhibitor, received US FDA approval for use in the treatment of metastatic CRPC in patients previously treated with docetaxel. Additionally, the novel androgen signaling inhibitor MDV3100 has been reported to confer a survival advantage compared to placebo in the same patient population. Here we review the scientific rationale for targeting androgen signaling in CRPC and the recent pivotal trials that support the use of novel second-line hormonal therapies. Additionally, we summarize ongoing preclinical and clinical efforts to ascertain and overcome mechanisms of resistance.. Novel inhibitors of extra-gonadal androgen synthesis and androgen receptor function demonstrate the continued importance of androgen signaling in CRPC. These agents have improved clinical outcomes for patients with metastatic CRPC.

Topics: Androgen Antagonists; Androgens; Androstadienes; Androstenes; Androstenols; Antineoplastic Agents; Benzamides; Benzimidazoles; Clinical Trials as Topic; Drug Resistance, Neoplasm; Humans; Male; Neoplasms, Hormone-Dependent; Nitriles; Phenylthiohydantoin; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Steroid 17-alpha-Hydroxylase; Thiohydantoins

Androgen receptor (AR)-mediated signaling is critical to the growth and survival of prostate cancer. Although medical castration and antiandrogen therapy can decrease AR activity and lower PSA, castration resistance eventually develops. Recent work exploring the molecular structure and evolution of AR in response to hormonal therapies has revealed novel mechanisms of progression of castration-resistant prostate cancer and yielded new targets for drug development. This review focuses on understanding the mechanisms of persistent AR signaling in the castrate environment, and highlights new therapies either currently available or in clinical trials, including androgen synthesis inhibitors and novel direct AR inhibitors.

Topics: Androgen Antagonists; Androstadienes; Antineoplastic Agents; Benzamides; Benzimidazoles; Humans; Imidazoles; Male; Naphthalenes; Nitriles; Phenylthiohydantoin; Prostatic Neoplasms; Receptors, Androgen; Steroid 17-alpha-Hydroxylase; Testosterone; Thiohydantoins

Galeterone, a novel prostate cancer candidate treatment, was discontinued after a Phase III clinical trial due to lack of efficacy. Galeterone is weakly basic and exhibits low solubility in biorelevant media (i.e., ~ 2 µg/mL in fasted simulated intestinal fluid). It was formulated as a 50-50 (w/w) galeterone-hypromellose acetate succinate spray-dried dispersion to increase its bioavailability. Despite this increase, the bioavailability of this formulation may have been insufficient and contributed to its clinical failure. We hypothesized that reformulating galeterone as an amorphous solid dispersion by KinetiSol® compounding could increase its bioavailability. In this study, we examined the effects of composition and manufacturing technology (Kinetisol and spray drying) on the performance of galeterone amorphous solid dispersions. KinetiSol compounding was utilized to create galeterone amorphous solid dispersions containing the complexing agent hydroxypropyl-β-cyclodextrin or hypromellose acetate succinate with lower drug loads that both achieved a ~ 6 × increase in dissolution performance versus the 50-50 spray-dried dispersion. When compared to a spray-dried dispersion with an equivalent drug load, the KinetiSol amorphous solid dispersions formulations exhibited ~ 2 × exposure in an in vivo rat study. Acid-base surface energy analysis showed that the equivalent composition of the KinetiSol amorphous solid dispersion formulation better protected the weakly basic galeterone from premature dissolution in acidic media and thereby reduced precipitation, inhibited recrystallization, and extended the extent of supersaturation during transit into neutral intestinal media.

Topics: Animals; Antineoplastic Agents; Biological Availability; Chemistry, Pharmaceutical; Drug Compounding; Humans; Male; Prostatic Neoplasms; Rats; Solubility; Spray Drying

We have investigated interactions of galeterone and its pharmacologically active metabolite - 3-keto-Δ4-galeterone (D4G) - with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2). It was shown by absorption spectroscopy that both compounds induce type I spectral changes of CYP21A2. Spectral dissociation constants (K

Topics: Androstadienes; Benzimidazoles; Drug Interactions; Enzyme Inhibitors; Humans; Male; Molecular Docking Simulation; Prostatic Neoplasms; Steroid 21-Hydroxylase

The androgen receptor (AR) is a steroid hormone receptor and its high expression and disruption of its regulation are strongly implicated in prostate cancer (PCa) development. One of the current therapies includes application of steroidal antiandrogens leading to blockade of the AR action by the abrogation of AR-mediated signaling. We introduced here novel 4,5,6,7-tetrahydropyrazolo[1,5-a]pyridine-fused steroidal compounds, described their synthesis based on [8π+2π] cycloaddition reactions of diazafulvenium methides with different steroidal scaffolds and showed their biological evaluation in different prostate cancer cell lines in vitro. Our results showed the ability of novel compounds to suppress the expression of known androgen receptor targets, Nkx3.1 and PSA in two prostate cell lines, 22Rv1 and VCaP. Candidate compound diminished the transcription of AR-regulated genes in the reporter cell line in a concentration-dependent manner. Antiproliferative activity of the most promising steroid was studied by clonogenic assay and induction of apoptosis in treated cells was documented by immunoblot detection of cleaved PARP.

Topics: Antineoplastic Agents; Binding Sites; Cell Line, Tumor; Homeodomain Proteins; Humans; Male; Molecular Docking Simulation; Prostatic Neoplasms; Pyrazoles; Pyridines; Receptors, Androgen; Steroids; Transcription Factors

Prostate cancer is one of the main causes of male cancer-related deaths worldwide and the suppression of androgen receptor signalling is established as an effective strategy for the treatment. A series of galeterone analogues including several steroid-fused azacycles, as well as 17-(benzimidazol-1-ylimino), 16α-(benzimidazol-2-ylamino), and 16α-(benzothiazol-2-ylamino) steroid derivatives, were synthesized and tested against prostate cancer cell lines. Candidate compound 3f was shown to reduce AR-regulated transcription in a dose-dependent manner in nanomolar ranges and suppress expression of AR-regulated proteins Nkx3.1 and PSA in 22Rv1-ARE14 and VCaP cancer cell lines. Flexible docking study revealed similar position of 3f within AR binding site in comparison of galeterone even with stronger binding energy.

Topics: Androstadienes; Antineoplastic Agents; Benzimidazoles; Cell Line; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Male; Molecular Structure; PC-3 Cells; Prostatic Neoplasms; Structure-Activity Relationship

According to the X-ray crystal structures of CYP17A1 (including its complexes with inhibitors), it is shown that a hydrogen bond exists between CYP17A1 and its inhibitors (such as abiraterone and TOK-001). Previous short MD simulations (50 ns) suggested that the binding of abiraterone to CYP17A1 is stronger than that of TOK-001. In this work, by carrying out long atomistic MD simulations (200 ns) of CYP17A1 and its complexes with abiraterone and TOK-001, we observed a binding mode between CYP17A1 and abiraterone, which is different from the binding mode between CYP17A1 and TOK-001. In the case of abiraterone binding, the unfilled volume in the active site cavity increases the freedom of movement of abiraterone within CYP17A1, leading to the collective motions of the helices G and B' as well as the breaking of hydrogen bond existing between the 3β-OH group of abiraterone and N202 of CYP17A1. However, the unfilled volume in the active site cavity can be occupied by the benzimidazole ring of TOK-001, restraining the motion of TOK-001. By pulling the two inhibitors (abiraterone and TOK-001) out of the binding pocket in CYP17A1, we discovered that abiraterone and TOK-001 were moved from their binding sites to the surface of protein similarly through the channels formed by the helices G and B'. In addition, based on the free energy calculations, one can see that it is energetically favorable for the two inhibitors (abiraterone and TOK-001) to enter into the binding pocket in CYP17A1.

Topics: Androstadienes; Androstenes; Antineoplastic Agents; Benzimidazoles; Binding Sites; Catalytic Domain; Cytochrome P-450 CYP1A1; Heme; Humans; Hydrophobic and Hydrophilic Interactions; Iron; Male; Molecular Docking Simulation; Prostatic Neoplasms; Protein Conformation, alpha-Helical

Galeterone is a steroidal CYP17A1 inhibitor, androgen receptor (AR) antagonist, and AR degrader, under evaluation in a phase III clinical trial for castration-resistant prostate cancer (CRPC). The A/B steroid ring (Δ

Topics: 17-Hydroxysteroid Dehydrogenases; 3-Oxo-5-alpha-Steroid 4-Dehydrogenase; Androstadienes; Animals; Benzimidazoles; Cell Line, Tumor; Chromatography, High Pressure Liquid; HEK293 Cells; Humans; Hydroxysteroid Dehydrogenases; Kaplan-Meier Estimate; Male; Mice; Pregnenolone; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Steroid 17-alpha-Hydroxylase; Tandem Mass Spectrometry; Transplantation, Heterologous

Topics: Androstadienes; Androstenedione; Aromatase Inhibitors; Benzimidazoles; Breast Neoplasms; Estrogens; Female; History, 20th Century; History, 21st Century; Humans; Male; Maryland; Prostatic Neoplasms

Topics: Androstadienes; Benzimidazoles; Humans; Male; Prostatic Neoplasms; Steroid 17-alpha-Hydroxylase

The androgen receptor (AR) is an important target for drug therapies combating prostate cancer. However, various acquired mutations within the AR sequence often render this receptor resistant to treatment. Ligand-induced interaction between the N- and C-termini of the AR marks the initial step in the AR signaling cascade and can thus serve as an early read-out for analysis of potential antagonists of wt and mutant AR. To measure changes of the N/C interaction in the wt and mutant AR variants upon the addition of inhibitors, we applied our recently developed Fluorescent Two-Hybrid (F2H) assay. The F2H method enables real-time monitoring and quantitative analysis of the interactions between GFP- and RFP-tagged proteins in live mammalian cells, where GFP-tagged proteins are tethered to a specific nuclear location. This anchoring approach provides a local signal enrichment suitable for direct visualization of protein-protein interactions as co-localizations by conventional epifluorescence microscopy. Since the F2H assay is fully reversible, we could monitor dynamics of AR N/C interactions in living cells in real time upon agonistic, as well as antagonistic treatments. In dose-response F2H experiments, we compared the potencies of abiraterone, bicalutamide, enzalutamide, flutamide, and galeterone/TOK-001 to prevent the dihydrotestosterone-induced N/C interaction in wt AR. We further applied the newly developed F2H assay to analyze how the AR N/C interaction is affected by the clinically relevant mutations W741L, F876L, T877A and F876L/T877A. We conclude that F2H is a reliable and technically undemanding approach for straightforward screening of new AR modulators, as well as for monitoring their activity in real time in living cells.

Topics: Androgen Antagonists; Androgens; Androstadienes; Androstenes; Anilides; Animals; Benzamides; Benzimidazoles; Biological Assay; Cell Line; Cricetinae; Dihydrotestosterone; Flutamide; HEK293 Cells; Humans; Male; Microscopy, Fluorescence; Mutation; Nitriles; Phenylthiohydantoin; Prostatic Neoplasms; Receptors, Androgen; Tosyl Compounds; Transcription Factors; Two-Hybrid System Techniques

The orteronel, abiraterone and galeterone, which were developed to treat castration resistant prostate cancer, inhibit 17,20 lyase activity but little is known about their effects on adrenal androgen biosynthesis. We studied the effect of several inhibitors and found that orteronel was selective towards 17,20 lyase activity than abiraterone and galeterone. Gene expression analysis showed that galeterone altered the expression of HSD3B2 but orteronel did not change the expression of HSD3B2, CYP17A1 and AKR1C3. The CYP19A1 activity was not inhibited except by compound IV which lowered activity by 23%. Surprisingly abiraterone caused complete blockade of CYP21A2 activity. Analysis of steroid metabolome by gas chromatography - mass spectrometry revealed changes in steroid levels caused by different inhibitors. We can conclude that orteronel is a highly specific inhibitor of 17,20 lyase activity. The discovery of these specific drug actions on steroidogenic enzyme activities would be valuable for understanding the regulation of androgens.

Topics: Adrenal Glands; Androgens; Androstadienes; Androstenes; Antineoplastic Agents; Benzimidazoles; Cell Line; Dose-Response Relationship, Drug; Humans; Imidazoles; Male; Naphthalenes; Prostatic Neoplasms; Steroid 17-alpha-Hydroxylase

Metastatic castration-resistant prostate cancer (mCRPC) accounts for a high percentage of prostate cancer mortality. The proprietary compound galeterone (gal) was designed to inhibit proliferation of androgen/androgen receptor (AR)-dependent prostate cancer cell in vitro and in vivo and is currently in phase III clinical development. Additionally, clinical studies with gal revealed its superb efficacy in four different cohorts of patients with mCRPC, including those expressing splice variant AR-V7. Preclinical studies with gal show that it also exhibits strong antiproliferative activities against AR-negative prostate cancer cells and tumors through a mechanism involving phosphorylation of eIF2α, which forms an integral component of the eukaryotic mRNA translation complex. Thus, we hypothesized that gal and its new analog, VNPT55, could modulate oncogenic mRNA translation and prostate cancer cell migration and invasion. We report that gal and VNPT55 profoundly inhibit migration and invasion of prostate cancer cells, possibly by down-regulating protein expression of several EMT markers (Snail, Slug, N-cadherin, vimentin, and MMP-2/-9) via antagonizing the Mnk-eIF4E axis. In addition, gal/VNPT55 inhibited both NF-κB and Twist1 transcriptional activities, down-regulating Snail and BMI-1 mRNA expression, respectively. Furthermore, profound up-regulation of E-cadherin mRNA and protein expression may explain the observed significant inhibition of prostate cancer cell migration and invasion. Moreover, expression of self-renewal proteins, β-catenin, CD44, and Nanog, was markedly depleted. Analysis of gal/VNPT55-treated CWR22Rv1 xenograft tissue sections also revealed that observations in vitro were recapitulated in vivo. Our results suggest that gal/VNPT55 could become promising agents for the prevention and/or treatment of all stages of prostate cancer.

Topics: Androstadienes; Animals; Benzimidazoles; Cell Line; Cell Movement; Eukaryotic Initiation Factor-4E; Gene Expression Regulation, Neoplastic; Humans; Immunoblotting; Intracellular Signaling Peptides and Proteins; Male; Mice, SCID; Neoplasm Invasiveness; NF-kappa B; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Xenograft Model Antitumor Assays

In our effort to discover potent and specific inhibitors of 17α-hydroxylase/17,20-lyase (CYP17), the key enzyme which catalyzes the biosynthesis of androgens from progestins, 3β-(hydroxy)-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (Galeterone or TOK-001, formerly called VN/124-1) was identified as a selective development candidate which modulates multiple targets in the androgen receptor (AR) signaling pathway. This drug annotation summarizes the mechanisms of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and human efficacy data for galeterone, which has successfully completed phase II clinical development in men with castration resistant (advanced) prostate cancer (CRPC). Phase III clinical studies in CRPC patients are scheduled to begin in early 2015.

Topics: Androgen Receptor Antagonists; Androstadienes; Benzimidazoles; Clinical Trials as Topic; Drug Discovery; Humans; Male; Molecular Structure; Molecular Targeted Therapy; Prostatic Neoplasms; Receptors, Androgen; Signal Transduction; Steroid 17-alpha-Hydroxylase; Tissue Distribution

Galeterone (Gal) is a first-in-class multi-target oral small molecule that will soon enter pivotal phase III clinical trials in castration resistant prostate cancer (CRPC) patients. Gal disrupts androgen receptor (AR) signaling via inhibition of CYP17, AR antagonism and AR degradation. Resistance to current therapy is attributed to up-regulation of full-length AR (fAR), splice variants AR (AR-Vs) and AR mutations. The effects of gal and VNPT55 were analyzed on f-AR and AR-Vs (AR-V7/ARv567es) in LNCaP, CWR22Rv1 and DU145 (transfected with AR-Vs) human PC cells in vitro and CRPC tumor xenografts. Galeterone/VNPT55 decreased fAR/AR-V7 mRNA levels and implicates Mdm2/CHIP enhanced ubiquitination of posttranslational modified receptors, targeting them for proteasomal degradation. Gal and VNPT55 also induced significant apoptosis in PC cells via increased Bax/Bcl2 ratio, cytochrome-c release with concomitant cleavage of caspase 3 and PARP. More importantly, gal and VNPT55 exhibited strong in vivo anti-CRPC activities, with no apparent host toxicities. This study demonstrate that gal and VNPT55 utilize cell-based mechanisms to deplete both fAR and AR-Vs. Importantly, the preclinical activity profiles, including profound apoptotic induction and inhibition of CRPC xenografts suggest that these agents offer considerable promise as new therapeutics for patients with CRPC and those resistant to current therapy.

Topics: Androstadienes; Animals; Antineoplastic Agents; Apoptosis; Benzimidazoles; Cell Line, Tumor; Cell Survival; Cytochromes c; Humans; Male; Mice; Mice, SCID; Neoplasm Transplantation; Phosphorylation; Poly(ADP-ribose) Polymerases; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Proteasome Endopeptidase Complex; Protein Isoforms; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-mdm2; Receptors, Androgen; RNA, Small Interfering

Galeterone inhibits the enzyme CYP17A1 and is currently in phase II clinical trials for castration-resistant prostate cancer (CRPC). Galeterone is also a direct androgen receptor (AR) antagonist and may enhance AR degradation. This study was undertaken to determine the molecular basis for AR effects and their therapeutic potential.. Effects of galeterone on AR expression and activities were examined in prostate cancer cell lines.. Similar to the AR antagonist enzalutamide, but in contrast to bicalutamide, galeterone did not induce binding of a constitutively active VP16-AR fusion protein to reporter genes and did not induce AR recruitment to endogenous androgen-regulated genes based on chromatin immunoprecipitation. Galeterone at low micromolar concentrations that did not induce cellular stress responses enhanced AR protein degradation in LNCaP and C4-2 cells, which express a T878A mutant AR, but not in prostate cancer cells expressing wild-type AR. Further transfection studies using stable LNCaP and PC3 cell lines ectopically expressing wild-type or T878A-mutant ARs confirmed that galeterone selectively enhances degradation of the T878A-mutant AR.. Similar to enzalutamide, galeterone may be effective as a direct AR antagonist in CRPC. It may be particularly effective against prostate cancer cells with the T878A AR mutation but may also enhance degradation of wild-type AR in vivo through a combination of direct and indirect mechanisms. Finally, these findings show that conformational changes in AR can markedly enhance its degradation and thereby support efforts to develop further antagonists that enhance AR degradation.

Topics: Androgen Receptor Antagonists; Androstadienes; Apoptosis; Benzimidazoles; Cell Proliferation; Chromatin; Chromatin Immunoprecipitation; Humans; Male; Mutant Proteins; Mutation; Prostatic Neoplasms; Prostatic Neoplasms, Castration-Resistant; Protein Binding; Proteolysis; Receptors, Androgen; Tumor Cells, Cultured

As part of our program to explore the influence of small structural modifications of our drug candidate 3β-(hydroxy)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (galeterone, 5) on the modulation of the androgen receptor (AR), we have prepared and evaluated a series of novel C-3, C-16, and C-17 analogues. Using structure activity analysis, we established that the benzimidazole moiety at C-17 is essential and optimal and also that hydrophilic and heteroaromatic groups at C-3 enhance both antiproliferative (AP) and AR degrading (ARD) activities. The most potent antiproliferative compounds were 3β-(1H-imidazole-1-carboxylate)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (47), 3-((EZ)-hydroximino)-17-(1H-benzimidazol-1-yl)androsta-4,16-diene (36), and 3β-(pyridine-4-carboxylate)-17-(1H-benzimidazol-1-yl)androsta-5,16-diene (43), with GI50 values of 0.87, 1.91, and 2.57 μM, respectively. Compared to 5, compound 47 was 4- and 8-fold more potent with respect to AP and ARD activities, respectively. Importantly, we also discovered that our compounds, including 5, 36, 43, and 47, could degrade both full-length and truncated ARs in CWR22rv1 human prostate cancer cells. With these activities, they have potential for development as new drugs for the treatment of all forms of prostate cancer.

Topics: Androstadienes; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Drug Design; Humans; Male; Molecular Targeted Therapy; Prostatic Neoplasms; Proteolysis; Receptors, Androgen; Steroid 17-alpha-Hydroxylase; Transcriptional Activation

TOK-001 and abiraterone are potent 17-heteroarylsteroid (17-HAS) inhibitors of Cyp17, one of the rate-limiting enzymes in the biosynthesis of testosterone from cholesterol in prostate cancer cells. Nevertheless, the molecular mechanism underlying the prevention of prostate cell growth by 17-HASs still remains elusive. Here, we assess the effects of 17-HASs on androgen receptor (AR) activity in LNCaP and LAPC-4 cells. We demonstrate that both TOK-001 and abiraterone reduced AR protein and mRNA expression, and antagonized AR-dependent promoter activation induced by androgen. TOK-001, but not abiraterone, is an effective apparent competitor of the radioligand [(3)H]R1881 for binding to the wild type and various mutant AR (W741C, W741L) proteins. In agreement with these data, TOK-001 is a consistently superior inhibitor than abiraterone of R1881-induced transcriptional activity of both wild type and mutant AR. However, neither agent was able to trans-activate the AR in the absence of R1881. Our data demonstrate that phospho-4EBP1 levels are significantly reduced by TOK-001 and to a lesser extent by abiraterone alcohol, and suggest a mechanism by which cap-dependent translation is suppressed by blocking assembly of the eIF4F and eIF4G complex to the mRNA 5' cap. Thus, the effects of these 17-HASs on AR signaling are complex, ranging from a decrease in testosterone production through the inhibition of Cyp17 as previously described, to directly reducing both AR protein expression and R1881-induced AR trans-activation.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Substitution; Androstadienes; Androstenes; Androstenols; Benzimidazoles; Cell Cycle Proteins; Cell Line, Tumor; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Male; Mutation, Missense; Phosphoproteins; Prostatic Neoplasms; Protein Binding; Receptors, Androgen; Steroid 17-alpha-Hydroxylase

Cytochrome P450 17A1 (also known as CYP17A1 and cytochrome P450c17) catalyses the biosynthesis of androgens in humans. As prostate cancer cells proliferate in response to androgen steroids, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer, but drug development has been hampered by lack of information regarding the structure of CYP17A1. Here we report X-ray crystal structures of CYP17A1, which were obtained in the presence of either abiraterone, a first-in-class steroidal inhibitor recently approved by the US Food and Drug Administration for late-stage prostate cancer, or TOK-001, an inhibitor that is currently undergoing clinical trials. Both of these inhibitors bind the haem iron, forming a 60° angle above the haem plane and packing against the central I helix with the 3β-OH interacting with aspargine 202 in the F helix. Notably, this binding mode differs substantially from those that are predicted by homology models and from steroids in other cytochrome P450 enzymes with known structures, and some features of this binding mode are more similar to steroid receptors. Whereas the overall structure of CYP17A1 provides a rationale for understanding many mutations that are found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate a better understanding of the enzyme's dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers.

Topics: Androstadienes; Androstenes; Androstenols; Antineoplastic Agents; Benzimidazoles; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Humans; Hydrogen Bonding; Ligands; Male; Models, Molecular; Prostatic Neoplasms; Protein Conformation; Receptors, Androgen; Steroid 17-alpha-Hydroxylase; United States; United States Food and Drug Administration

Prostate Cancer (PCa), a leading cause of cancer death worldwide (www.cancer.gov), is a complex malignancy where a spectrum of targets leads to a diversity of PCa forms. A widely pursued therapeutic target is the Androgen Receptor (AR). As a Steroid Hormone Receptor, AR serves as activator of transcription upon binding to androgens and plays a central role in the development of PCa. AR is a structurally flexible protein, and conformational plasticity of residues in the binding-pocket is a key to its ability to accommodate ligands from various chemical classes. Besides direct modulation of AR activity by antagonists, inhibition of cytochrome CYP17 (17α-hydroxylase/17,20-lyase), essential in androgen biosynthesis, has widely been considered an effective strategy against PCa. Interestingly, Handratta et al. (2005) discovered new, potent inhibitors of CYP17 (C-17 steroid derivatives) with pure AR antagonistic properties. Although the antiandrogenic activity of their lead compound (VN/124-1) has been experimentally proven both in vitro and in vivo, no structural data are currently available to elucidate the molecular determinants responsible for these desirable dual inhibitory properties. We implemented a Structure-based Drug Design (SBDD) approach to generate a valuable hypothesis as to the binding modes of steroidal CYP17 inhibitors/antiandrogens against the AR. To deal with the plasticity of residues buried in the Ligand Binding Domain (LBD), we developed a flexible-receptor Docking protocol based on Induced-Fit (IFD) methodology (www.schrodinger.com/). Our results constitute an ideal starting point for the rational design of next-generation analogues of CYP17 inhibitors/antiandrogens as well as an attractive tool to suggest novel chemical classes of AR antagonists.

Topics: Androgen Receptor Antagonists; Androstadienes; Benzimidazoles; Binding Sites; Cell Line, Tumor; Clinical Trials as Topic; Crystallography, X-Ray; Drug Design; Humans; Male; Molecular Docking Simulation; Mutation; Prostatic Neoplasms; Protein Binding; Protein Structure, Secondary; Receptors, Androgen; Steroid 17-alpha-Hydroxylase; Structure-Activity Relationship; Testosterone; Thermodynamics

In a continuing study of our clinical candidate 5 VN/124-1 (TOK-001) and analogs as potential agents for prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using western blot analysis, we have generated a preliminary structure-activity relationship (SAR) of 5 and related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical trials. In our desire to optimize the potency of 5, compounds 6 (3ξ-fluoro-) and 9 (3β-sulfamate-) designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We showed, that on equimolar basis, compound 6 was ∼2-fold more efficacious versus LAPC-4 xenografts than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a potential treatment of prostate cancer.

Topics: Androstadienes; Androstenes; Androstenols; Animals; Antineoplastic Agents; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Clinical Trials, Phase III as Topic; Drug Combinations; Estradiol; Humans; Male; Mice; Mice, SCID; Norethindrone; Prostatic Neoplasms; Receptors, Androgen; Steroid 17-alpha-Hydroxylase; Structure-Activity Relationship; Testosterone; Xenograft Model Antitumor Assays

To determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer (PCa), we investigated the relationship between the androgen receptor (AR) and mTOR pathways and the impact of inhibiting both pathways in androgen-dependent and castration-resistant PCa models.. Androgen-dependent (LNCaP) and castration-resistant PCa (HP-LNCaP) cells were grown as tumours in SCID mice. Once tumours reached 500 mm(3), animals were grouped and injected subcutaneous with vehicle, our novel anti-androgen/androgen synthesis inhibitor, VN/124-1, bicalutamide, and everolimus. Tumour volumes were measured biweekly. The PSA and protein analyses were performed after completion of the treatment.. The addition of everolimus to bicalutamide treatment of resistant tumours significantly reduced tumour growth rates and tumour volumes. Anti-androgen treatment also increased protein expression of multiple signal transduction pathways earlier than vehicle-treated control xenografts. VN/124-1 plus everolimus acted in concert to reduce tumour growth rates in our castration-resistant xenograft model.. This study suggests that dual inhibition of AR and mTOR in castration-resistant xenograft models can restore sensitivity of tumours to anti-androgen therapy. Furthermore, after bicalutamide failure, dual inhibition with VN/124-1 and everolimus was the most effective treatment.

Topics: Androgen Antagonists; Androgen Receptor Antagonists; Androstadienes; Anilides; Animals; Benzimidazoles; Castration; Cell Line, Tumor; Disease Progression; Drug Therapy, Combination; Everolimus; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, SCID; Neoplasms, Hormone-Dependent; Nitriles; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Receptor Cross-Talk; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tosyl Compounds; Xenograft Model Antitumor Assays

We previously reported that our novel compound 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (VN/124-1) is a potent 17alpha-hydroxylase/17,20-lyase (CYP17) inhibitor/antiandrogen and strongly inhibits the formation and proliferation of human prostate cancer LAPC4 tumor xenografts in severe combined immunodeficient mice. In this study, we report that VN/124-1 and other novel CYP17 inhibitors also cause down-regulation of androgen receptor (AR) protein expression in vitro and in vivo. This mechanism of action seems to contribute to their antitumor efficacy. We compared the in vivo antitumor efficacy of VN/124-1 with that of castration and a clinically used antiandrogen, Casodex, and show that VN/124-1 is more potent than castration in the LAPC4 xenograft model. Treatment with VN/124-1 (0.13 mmol/kg twice daily) was also very effective in preventing the formation of LAPC4 tumors (6.94 versus 2410.28 mm(3) in control group). VN/124-1 (0.13 mmol/kg twice daily) and VN/124-1 (0.13 mmol/kg twice daily) + castration induced regression of LAPC4 tumor xenografts by 26.55% and 60.67%, respectively. Treatments with Casodex (0.13 mmol/kg twice daily) or castration caused significant tumor suppression compared with control. Furthermore, treatment with VN/124-1 caused marked down-regulation of AR protein expression, in contrast to treatments with Casodex or castration that caused significant up-regulation of AR protein expression. The results suggest that VN/124-1 acts by several mechanisms (CYP17 inhibition, competitive inhibition, and down-regulation of the AR). These actions contribute to inhibition of the formation of LAPC4 tumors and cause regression of growth of established tumors. VN/124-1 is more efficacious than castration in the LAPC4 xenograft model, suggesting that the compound has potential for the treatment of prostate cancer.

Topics: Androgen Receptor Antagonists; Androstadienes; Antineoplastic Agents; Benzimidazoles; Binding, Competitive; Cell Line, Tumor; Down-Regulation; Enzyme Inhibitors; Humans; Male; Prostatic Neoplasms; Receptors, Androgen; Steroid 17-alpha-Hydroxylase

Inhibitors of the enzyme 17alpha-hydroxylase/17,20 lyase are a new class of anti-prostate cancer agents currently undergoing preclinical and clinical development. We have previously reported the superior anticancer activity of our novel 17alpha-hydroxylase/17,20 lyase inhibitor, VN/124-1, against androgen-dependent cancer models. Here, we examined the effect of VN/124-1 on the growth of the androgen-independent cell lines PC-3 and DU-145 and found that the compound inhibits their growth in a dose-dependent manner in vitro (GI50, 7.82 micromol/L and 7.55 micromol/L, respectively). We explored the mechanism of action of VN/124-1 in PC-3 cells through microarray analysis and found that VN/124-1 up-regulated genes involved in stress response and protein metabolism, as well as down-regulated genes involved in cell cycle progression. Follow-up real-time PCR and Western blot analyses revealed that VN/124-1 induces the endoplasmic reticulum stress response resulting in down-regulation of cyclin D1 protein expression and cyclin E2 mRNA. Cell cycle analysis confirmed G1-G0 phase arrest. Measurements of intracellular calcium levels ([Ca2+]i) showed that 20 micromol/L VN/124-1 caused a release of Ca2+ from endoplasmic reticulum stores resulting in a sustained increase in [Ca2+]i. Finally, cotreatment of PC-3 cells with 5, 10, and 20 micromol/L VN/124-1 with 10 nmol/L thapsigargin revealed a synergistic relationship between the compounds in inhibiting PC-3 cell growth. Taken together, these findings show VN/124-1 is endowed with multiple anticancer properties that may contribute to its utility as a prostate cancer therapeutic.

Topics: Androgens; Androstadienes; Animals; Benzimidazoles; Calcium; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Down-Regulation; Drug Synergism; Endoplasmic Reticulum; Eukaryotic Initiation Factor-2; G1 Phase; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Male; Oligonucleotide Array Sequence Analysis; Phosphorylation; Prostatic Neoplasms; Resting Phase, Cell Cycle; Steroid 17-alpha-Hydroxylase; Thapsigargin; Up-Regulation

This study was carried out to determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer. We investigated the role of the androgen receptor and its relationship to other signal transduction proteins. A hormone-refractory prostate cancer cell line [high-passage LNCaP (HP-LNCaP)] was established in vitro. Cells were treated with inhibitors of mammalian target of rapamycin and tyrosine kinase receptors. Expression of these proteins and the androgen receptor were measured by Western immunoblotting. Analysis of the model and various treatments was also assessed through proliferation assays, luciferase activation assays, binding assays, and ELISA. Our novel antiandrogen, VN/124-1, effectively inhibited proliferation of hormone-resistant prostate cancer cell lines (HP-LNCaP), which were no longer sensitive to bicalutamide and had increased expression of the androgen receptor. Treatment with everolimus or gefitinib resulted in an increase in protein expression and activation of the androgen receptor. Conversely, inhibition of the androgen receptor resulted in increased expression of IGFR1beta, pHER2, pmTOR, and pAkt. The addition of bicalutamide to everolimus or gefitinib inhibited cell proliferation in HP-LNCaP cells. However, the addition of VN/124-1 has proven to be superior to bicalutamide, and the combination was synergistic (P<0.05) compared with either agent alone. This study suggests that compensatory cross-talk between the androgen receptor and various signaling pathways may account for decreased sensitivity to androgen receptor antagonists and the progression to hormone-resistant prostate cancer. Furthermore, these findings suggest that inhibition of both pathways may provide effective control in hormone-resistant prostate cancer and restore sensitivity to androgen antagonists in hormone-refractory patients.

Topics: Androgen Antagonists; Androgens; Androstadienes; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Disease Progression; Drug Resistance, Neoplasm; Humans; Male; Models, Biological; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Binding; Signal Transduction

New chemical entities, steroidal C-17 benzoazoles (5, 6, 9 and 10) and pyrazines (14 and 15) were rationally designed and synthesized. The key reaction for synthesis of the benzoazoles involved the nucleophilic vinylic "addition-elimination" substitution reaction of 3beta-acetoxy-17-chloro-16-formylandrosta-5,16-diene (2) and benzoazole nucleophiles, while that for synthesis of pyrazines involved palladium-catalyzed cross-coupling reaction of 17-iodoandrosta-5,16-dien-3beta-ol (13) with tributylstannyl diazines. Some of the compounds were shown to be potent inhibitors of human CYP17 enzyme as well as potent antagonist of both wild type and mutant androgen receptors (AR). The most potent CYP17 inhibitors were 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (5, code named VN/124-1), 3beta-hydroxy-17-(5(1)-pyrimidyl)androsta-5,16-diene (15) and 17-(1H-benzimidazole-1-yl)androsta-4,16-dien-3-one (6), with IC(50) values of 300, 500 and 915 nM, respectively. Compounds 5, 6, 14 and 15 were effective at preventing binding of (3)H-R1881 (methyltrienolone, a stable synthetic androgen) to both the mutant LNCaP AR and the wild-type AR, but with a 2.2- to 5-fold higher binding efficiency to the latter. Compounds 5 and 6 were also shown to be potent pure AR antagonists. The cell growth studies showed that 5 and 6 inhibit the growth of DHT-stimulated LNCaP and LAPC4 prostate cancer cells with IC(50) values in the low micromolar range (i.e., <10 microM). Their inhibitory potencies were comparable to that of casodex but remarkably superior to that of flutamide. The pharmacokinetics of compounds 5 and 6 in mice were investigated. Following s.c. administration of 50 mg/kg of 5 and 6, peak plasma levels of 16.82 and 5.15 ng/mL, respectively, occurred after 30 to 60 min, both compounds were cleared rapidly from plasma (terminal half-lives of 44.17 and 39.93 min, respectively), and neither was detectable at 8 h. Remarkably, compound 5 was rapidly converted into a metabolite tentatively identified as 17-(1H-benzimidazol-1-yl)androsta-3-one. When tested in vivo, 5 proved to be very effective at inhibiting the growth of androgen-dependent LAPC4 human prostate tumor xenograft, while 6 was ineffective. Compound 5 (50 mg/kg/twice daily) resulted in a 93.8% reduction (P = 0.00065) in the mean final tumor volume compared with controls, and it was also significantly more effective than castration. To our knowledge, this is the first example of an antihormonal agent (an inhi

Topics: 5-alpha Reductase Inhibitors; Androgen Antagonists; Androstadienes; Animals; Antineoplastic Agents; Azoles; Benzimidazoles; Cell Line, Tumor; Cell Proliferation; Humans; Isoenzymes; Male; Mice; Mice, SCID; Mutation; Prostatic Neoplasms; Pyrazines; Radioligand Assay; Receptors, Androgen; Steroid 17-alpha-Hydroxylase; Structure-Activity Relationship; Tissue Distribution; Transcription, Genetic; Transcriptional Activation; Xenograft Model Antitumor Assays