Salinization Dramatically Enhance the Anti-Prostate Cancer Efficacies of AR/AR-V7 and Mnk1/2 Molecular Glue Degraders, Galeterone and VNPP433-3ß Which Outperform Docetaxel and Enzalutamide in CRPC CWR22Rv1 Xenograft Mouse Model.

Galeterone, 3ß-(hydroxy)-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (Gal, 1) and VNPP433-3ß, 3ß-(1H-imidazole-1-yl-17-(1H-benzimidazole-1-yl)androsta-5,16-diene (2) are potent molecular glue degrader modulators of AR/AR-V7 and Mnk1/2-eIF4E signaling pathways, and are promising Phase 3 and Phase 1 drug candidates, respectively. Because appropriate salts can be utilized to create new chemical entities with enhanced aqueous solubility, in vivo pharmacokinetics, and enhanced in vitro and in vivo efficacies, the monohydrochloride salt of Gal (3) and the mono- and di-hydrochlorides salts of compound 2, compounds 4 and 5, respectively, were synthesized. The salts were characterized using 1H NMR, 13C NMR and HRMS analyses. Compound 3 displayed enhanced in vitro antiproliferative activity (7.4-fold) against three prostate cancer cell lines but surprisingly decreased plasma exposure in the pharmacokinetics study. The antiproliferative activities of the compound 2 salts (4 and 5) were equivalent to that of compound 2, but their oral pharmacokinetic profiles were significantly enhanced. Finally, and most importantly, oral administration of the parent compounds (1 and 2) and their corresponding salts (3, 4 and 5) caused dose-dependent potent inhibition/regression of aggressive and difficult-to-treat CWR22Rv1 tumor xenografts growth, with no apparent host toxicities and were highly more efficacious than the blockbuster FDA-approved prostate cancer drugs, Enzalutamide (Xtandi) and Docetaxel (Taxotere). Thus, the HCl salts of Gal (3) and VNPP433-3ß (4 and 5) are excellent orally bioavailable candidates for clinical development.

Transcriptome profiling reveals that VNPP433-3ß, the lead next-generation galeterone analog inhibits prostate cancer stem cells by downregulating epithelial-mesenchymal transition and stem cell markers.

Cancer stem cells (CSCs) virtually present in all tumors albeit in small numbers are primarily responsible for driving cancer progression, metastasis, drug resistance, and recurrence. Prostate cancer (PCa) is the second most frequent cancer in men worldwide, and castration resistant prostate cancer (CRPC) remains a major challenge despite the tremendous advancements in medicine. Currently, none of the available treatment options are effective in treating CRPC. We earlier reported that VNPP433-3ß, the lead next-generation galeterone analog is effective in treating preclinical in vivo models of CRPC. In this study using RNA-seq, cytological, and biochemical methods, we report that VNPP433-3ß inhibits prostate CSCs by targeting key pathways critical to stemness and epithelial-mesenchymal transition. VNPP433-3ß inhibits CSCs in PCa, presumably by degrading the androgen receptor (AR) thereby decreasing the AR-mediated transcription of several stem cell markers including BMI1 and KLF4. Transcriptome analyses by RNA-seq, Ingenuity Pathway Analysis, and Gene Set Enrichment Analysis demonstrate that VNPP433-3ß inhibits transcription of several genes and functional pathways critical to the prostate CSCs thereby inhibiting CSCs in PCa besides targeting the bulk of the tumor.

Targeting of protein translation as a new treatment paradigm for prostate cancer.

PURPOSE OF REVIEW: The current overview will summarize some of the developments in the area of protein translation, including their relation to the therapeutic targeting of prostate cancer. RECENT FINDINGS: Translational control, mediated by the rate-limiting eukaryotic translation initiation factor 4E (eIF4E), drives selective translation of several oncogenic proteins, thereby contributing to tumor growth, metastasis, and treatment resistance in various cancers, including prostate cancer. As an essential regulatory hub, several oncogenic hyperactive signaling pathways appear to converge on eIF4E to promote tumorigenesis. Several approaches that target the eIF4E-dependent protein translation network are being actively studied, and it is likely that some may ultimately emerge as promising anticancer therapeutics. SUMMARY: An array of inhibitors has shown promise in targeting specific components of the translational machinery in several preclinical models of prostate cancer. It is hoped that some of these approaches may ultimately have relevance in improving the clinical outcomes of patients with advanced prostate cancer.

Regulation of androgen receptor activity by tyrosine phosphorylation.

The androgen receptor (AR) is essential for the growth of prostate cancer cells. Here, we report that tyrosine phosphorylation of AR is induced by growth factors and elevated in hormone-refractory prostate tumors. Mutation of the major tyrosine phosphorylation site in AR significantly inhibits the growth of prostate cancer cells under androgen-depleted conditions. The Src tyrosine kinase appears to be responsible for phosphorylating AR, and there is a positive correlation of AR tyrosine phosphorylation with Src tyrosine kinase activity in human prostate tumors. Our data collectively suggest that growth factors and their downstream tyrosine kinases, which are elevated during hormone-ablation therapy, can induce tyrosine phosphorylation of AR and such modification may be important for prostate tumor growth under androgen-depleted conditions.

Sterol-like drugs potentiate statin-triggered prostate cancer cell death by inhibiting SREBP2 nuclear translocation.

There is an urgent need to provide immediate and effective options for the treatment of prostate cancer (PCa) to prevent progression to lethal castration-resistant PCa (CRPC). The mevalonate (MVA) pathway is dysregulated in PCa, and statin drugs commonly prescribed for hypercholesterolemia, effectively target this pathway. Statins exhibit anti-PCa activity, however the resulting intracellular depletion of cholesterol triggers a feedback loop that restores MVA pathway activity, thus diminishing statin efficacy and contributing to resistance. To identify drugs that block this feedback response and enhance the pro-apoptotic activity of statins, we performed a high-content image-based screen of a 1508 drug library, enriched for FDA-approved compounds. Two of the validated hits, Galeterone (GAL) and Quinestrol, share the cholesterol-related tetracyclic structure, which is also evident in the FDA-approved CRPC drug Abiraterone (ABI). Molecular modeling revealed that GAL, Quinestrol and ABI not only share structural similarity with 25-hydroxy-cholesterol (25HC) but were also predicted to bind similarly to a known protein-binding site of 25HC. This suggested GAL, Quinestrol and ABI are sterol-mimetics and thereby inhibit the statin-induced feedback response. Cell-based assays demonstrated that these agents inhibit nuclear translocation of sterol-regulatory element binding protein 2 (SREBP2) and the transcription of MVA genes. Sensitivity was independent of androgen status and the Fluva-GAL combination significantly impeded CRPC tumor xenograft growth. By identifying cholesterol-mimetic drugs that inhibit SREBP2 activation upon statin treatment, we provide a potent "one-two punch" against CRPC progression and pave the way for innovative therapeutic strategies to combat additional diseases whose etiology is associated with SREBP2 dysregulation.

The combination of the histone deacetylase inhibitor vorinostat and synthetic triterpenoids reduces tumorigenesis in mouse models of cancer.

Novel drugs and drug combinations are needed for the chemoprevention and treatment of cancer. We show that the histone deacetylase inhibitor vorinostat [suberoylanilide hydroxamic acid (SAHA)] and the methyl ester or ethyl amide derivatives of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO-Me and CDDO-Ea, respectively) cooperated to inhibit the de novo synthesis of nitric oxide in RAW 264.7 macrophage-like cells and in primary mouse peritoneal macrophages. Additionally, SAHA enhanced the ability of synthetic triterpenoids to delay formation of estrogen receptor-negative mammary tumors in MMTV-polyoma middle T (PyMT) mice. CDDO-Me (50 mg/kg diet) and SAHA (250 mg/kg diet) each significantly delayed the initial development of tumors by 4 (P < 0.001) and 2 (P < 0.05) weeks, respectively, compared with the control group in the time required to reach 50% tumor incidence. CDDO-Ea (400 mg/kg diet), as a single agent, did not delay tumor development. The combination of either triterpenoid with SAHA was significantly more potent than the individual drugs for delaying tumor development, with a 7 week (P < 0.001) delay before 50% tumor incidence was reached. SAHA, alone and in combination with CDDO-Me, also significantly (P < 0.05) inhibited the infiltration of tumor-associated macrophages into the mammary glands of PyMT mice and levels of the chemokine macrophage colony-stimulating factor in primary PyMT tumor cells. In addition, SAHA and the synthetic triterpenoids cooperated to suppress secreted levels of the pro-angiogenic factor matrix metalloproteinase-9. Similar results were observed in mouse models of pancreatic and lung cancer. At concentrations that were anti-inflammatory, SAHA had no effect on histone acetylation. These studies suggest that both SAHA and triterpenoids effectively delay tumorigenesis, thereby demonstrating a promising, novel drug combination for chemoprevention.

Targeted Degradation of Androgen Receptor by VNPP433-3ß in Castration-Resistant Prostate Cancer Cells Implicates Interaction with E3 Ligase MDM2 Resulting in Ubiquitin-Proteasomal Degradation.

Targeted protein degradation is a fast-evolving therapeutic strategy to target even the traditionally undruggable target proteins. Contrary to the traditional small-molecule inhibitors of enzyme or receptor antagonists that bind the active site pockets in the target protein, molecular glue degraders facilitate interaction of target proteins with E3 ubiquitin ligases by stabilizing the ternary complex and induce physical proximity, thereby triggering ubiquitination and subsequent proteasomal degradation. AR plays a key role in all stages of prostate cancer. It is activated by the binding of androgenic hormones and transcriptionally regulates multiple genes including the ones that regulate cell cycle. Using HiBiT CRISPR cell line, biochemical methods, and RNA sequencing, we report the potential role of VNPP433-3ß, the next generation galeterone analog as molecular glue that brings together AR, the key driver of prostate cancer and MDM2, an E3 ubiquitin ligase leading to ubiquitination and subsequent degradation of f-AR and AR-V7 in prostate cancer cells.

Murine toxicology and pharmacokinetics of lead next generation galeterone analog, VNPP433-3ß.

VNPP433-3ß (compound 2, (3ß-(1H-imidazole-1-yl)-17-(1H-benzimidazole-1-yl)-androsta-5,16-diene), a multitarget anticancer agent has emerged as our lead next generation galeterone analogs (NGGA). Compound 2 is currently in development as potential new therapeutic for prostate and pancreatic cancers. The preliminary toxicity study reveals that the compound 2 was better tolerated by the normal male CD-1 mice than the male Nude mice. The maximum tolerated dose (MTD) in the Nude mice was estimated to be between 25 < 50 mg/kg. After oral dosing of compound 2 to male and female rats, the plasma concentration versus time curves were very consistent between animals and the AUClast increased with dose. Many plasmas concentration versus time curves profiles were nearly flat over 24 hr., suggesting extended absorption from the GI tract. Consequently, reliable values for half-life and AUCinf were not determined. Calculated oral bioavailability (using oral AUClast and excluding the outlier IV animal) ranged from 32 to 47 %. This should be considered a minimum value since the contribution to true AUC beyond 24 hr. is clearly not zero. Clearly, these toxicology and pharmacokinetics parameters pave the way for understanding the anticancer pharmacological actions and provide a meaningful basis for further preclinical development and eventual clinical development.

VNLG-152R and its deuterated analogs potently inhibit/repress triple/quadruple negative breast cancer of diverse racial origins in vitro and in vivo by upregulating E3 Ligase Synoviolin 1 (SYVN1) and inducing proteasomal degradation of MNK1/2.

Triple-negative breast cancer (TNBC) and its recently identified subtype, quadruple negative breast cancer (QNBC), collectively account for approximately 13% of reported breast cancer cases in the United States. These aggressive forms of breast cancer are associated with poor prognoses, limited treatment options, and lower overall survival rates. In previous studies, our research demonstrated that VNLG-152R exhibits inhibitory effects on TNBC cells both in vitro and in vivo and the deuterated analogs were more potent inhibitors of TNBC cells in vitro. Building upon these findings, our current study delves into the molecular mechanisms underlying this inhibitory action. Through transcriptome and proteome analyses, we discovered that VNLG-152R upregulates the expression of E3 ligase Synoviolin 1 (SYVN1), also called 3-hydroxy-3-methylglutaryl reductase degradation (HRD1) in TNBC cells. Moreover, we provide genetic and pharmacological evidence to demonstrate that SYVN1 mediates the ubiquitination and subsequent proteasomal degradation of MNK1/2, the only known kinases responsible for phosphorylating eIF4E. Phosphorylation of eIF4E being a rate-limiting step in the formation of the eIF4F translation initiation complex, the degradation of MNK1/2 by VNLG-152R and its analogs impedes dysregulated translation in TNBC cells, resulting in the inhibition of tumor growth. Importantly, our findings were validated in vivo using TNBC xenograft models derived from MDA-MB-231, MDA-MB-468, and MDA-MB-453 cell lines, representing different racial origins and genetic backgrounds. These xenograft models, which encompass TNBCs with varying androgen receptor (AR) expression levels, were effectively inhibited by oral administration of VNLG-152R and its deuterated analogs in NRG mice. Importantly, in direct comparison, our compounds are more effective than enzalutamide and docetaxel in achieving tumor growth inhibition/repression in the AR+ MDA-MD-453 xenograft model in mice. Collectively, our study sheds light on the involvement of SYVN1 E3 ligase in the VNLG-152R-induced degradation of MNK1/2 and the therapeutic potential of VNLG-152R and its more potent deuterated analogs as promising agents for the treatment of TNBC across diverse patient populations.

Anti-tumor effects of a novel retinoic acid metabolism blocking agent VN/14-1 in the N-methyl-N-nitrosourea-induced rat mammary carcinoma model and its effects on the uterus.

VN/14-1 [4-(±)-(1H-Imidazol-1-yl)-(E)-retinoic acid], a novel retinoic acid metabolism blocking agent (RAMBA), works by inhibiting the breakdown of all-trans-retinoic acid. The purpose of this study was to evaluate the anti-tumor effects of VN/14-1 on the N-methyl-N-nitrosourea (MNU)-induced rat mammary carcinoma model, and peripheral organ effects on the uteri of immature ovariectomized (OVX) rats. In tumor burden experiments, after 56 days of administration of VN/14-1 5, 10, and 20 mg/kg/day, significant tumor reductions in mean tumor weight of 19.1, 34.4, and 44.3%, compared to tumors in control animals occurred. Cumulative tumor growth was also significantly slower in a dose-dependent manner in groups receiving 5, 10, and 20 mg/kg/day of VN/14-1 compared to growth rates in the control group. Tumor apoptosis was significant increases in animals treated with 5, 10, and 20 mg/kg/day of VN/14-1. In uterotrophic experiments, immature OVX rats given VN/14-1 significantly reduced uterine weight and blocked endometrial stimulation induced by unopposed ß-estradiol (E2). In both rat models, adverse toxicities included weakness, anorexia, and reduction in body weight in the groups given the highest dose of 20 mg/kg/day. In summary, VN/14-1 inhibited tumor growth in the MNU-induced estrogen receptor (ER)-positive rat mammary tumor model, and antagonized the stimulatory effect of estrogens on the uterus. The studies suggest that VN/14-1 may be a useful novel therapy for ER-positive breast cancer.

Novel deuterated Mnk1/2 protein degrader VNLG-152R analogs: Synthesis, In vitro Anti-TNBC activities and pharmacokinetics in mice.

A new and improved synthesis of lead Mnk1/2 protein degrader, VNLG-152R, 4-(±)-(1H-imidazole-1-yl)-N-(4-fluorophenyl)-(E)-retinamide (1) has been developed from commercially available 4-oxo-ATRA (8). This procedure was also utilized to synthesize the seven possible deuterated analogs of compound 1 (11-17). The deuterated analogs were either better or equipotent to 1 in in vitro antiproliferative activities against MDA-MB-231 and MDA-MB-468 human TNBC cells. The Mnk1/2 degraders were equally effective as a standard TNBC therapy (paclitaxel). Importantly, the expression of Mnk1, peIF4E and their associated downstream targets, including cyclin D1 and Bcl2, were strongly decreased in compound 1/analogs (11-17)-treated TNBC cells signifying inhibition of Mnk1-eIF4E signaling. More importantly, we showed that deuterated analogs, 12, 16 and 17 possess improved pharmacokinetics parameters following oral administration to CD-1 female mice compared to the parent non-deuterated compound 1, thus addressing the rapid clearance (short half-life and short residence time) pharmacokinetic inadequacy of compound 1.

Large-scale synthesis of galeterone and lead next generation galeterone analog VNPP433-3ß.

VNPP433-3ß (compound 2, (3ß-(1H-imidazole-1-yl)-17-(1H-benzimidazole-1-yl)-androsta-5,16-diene), a multitarget anticancer agent has emerged as our lead next generation galeterone analogs (NGGA). Here, we describe a large multi-gram (92 g) scale synthesis of compound 2 starting from the commercially available dehydroepiandrosterone-3-acetate (DHEA, 6) via Galeterone (Gal, 1), in 8 steps with a 26% overall yield and 99.5% purity. The overall yield for the synthesis of Gal from DHEA improved from previously reported 47% to 59%. The advantages of this synthesis are as follows: (1) In the first two steps of Scheme 2, the change of solvents and reagents enabled the isolation of compounds 7 and 8 from heptane triturations, as column chromatography was eliminated in both steps. (2) In step 3 (deformylation) the catalyst required was reduced from 50% to 10% (wt/wt) of compound 8 which enable easy purification of compound 9, with modest increased yield. (3) The fourth step to produce Gal (1) was improved by using methanol, eliminating the use of tetrahydrofuran (THF) and dichloromethane, solvent which may be a problem as residual solvent contaminant. (4) In the final step 8, the imidazole-ring formation, inexpensive glyoxal (40% aqueous solution) was used in the reaction instead of expensive glyoxal trimer dihydrate. The structure of the target product (2, VNPP433-3ß) was established by NMR spectroscopy, mass spectrometry and elemental analysis. Gal and VNPP433-3ß exhibit more potent antiproliferative activities against CWR22Rv1 human prostate cancer cells compared to clinical drugs, Abiraterone and Enzalutamide.

Novel AR/AR-V7 and Mnk1/2 Degrader, VNPP433-3ß: Molecular Mechanisms of Action and Efficacy in AR-Overexpressing Castration Resistant Prostate Cancer In Vitro and In Vivo Models.

Prostate cancer (PCa) relies in part on AR-signaling for disease development and progression. Earlier, we developed drug candidate galeterone, which advanced through phase 2-clinical trials in treating castration-resistant PCa (CRPC). Subsequently, we designed, synthesized, and evaluated next-generation galeterone-analogs including VNPP433-3ß which is potently efficacious against pre-clinical models of PCa. This study describes the mechanism of action of VNPP433-3ß that promotes degradation of full-length AR (fAR) and its splice variant AR-V7 besides depleting MNK1/2 in in vitro and in vivo CRPC models that stably overexpresses fAR. VNPP433-3ß directly engages AR within the cell and promotes proteasomal degradation of fAR and its splice variant AR-V7 by enhancing the interaction of AR with E3 ligases MDM2/CHIP but disrupting AR-HSP90 binding. Next, VNPP433-3ß decreases phosphorylation of 4EBP1 and abates binding of eIF4E and eIF4G to 5' cap of mRNA by depleting MNK1/2 with consequent depletion of phosphorylated eIF4E. Finally, RNA-seq demonstrates modulation of multiple pathways that synergistically contribute to PCa inhibition. Therefore, VNPP433-3ß exerts its antitumor effect by imposing 1) transcriptional regulation of AR and AR-responsive oncogenes 2) translational regulation by disrupting mRNA-5'cap-dependent translation initiation, 3) reducing AR half-life through enhanced proteasomal degradation in vitro and AR-overexpressing tumor xenografts in vivo.

Targeting drug-metabolizing enzymes for effective chemoprevention and chemotherapy.

The primary focus of chemoprevention research is the prevention of cancer using pharmacological, biological, and nutritional interventions. Chemotherapeutic approaches that have been used successfully for both the prevention and treatment of a number of human malignancies have arisen from the identification of specific agents and appropriate molecular targets. Although drug-metabolizing enzymes have historically been targeted in attempts to block the initial, genotoxic events associated with the carcinogenic process, emerging evidence supports the idea that manipulating drug-metabolizing enzymes may also be an effective strategy to be used for treating tumor progression, invasion, and, perhaps, metastasis. This report summarizes a symposium that presents some recent progress in this area. One area of emphasis is the development of a CYP17 inhibitor for treatment of prostate cancer that may also have androgen-independent anticancer activity at higher concentrations. A second focus is the use of a mouse model to investigate the effects of aryl hydrocarbon receptor and Cyp1b1 status and chemopreventative agents on transplacental cancer. A third area of focus is the phytochemical manipulation of not only cytochrome P450 (P450) enzymes but also phase II inflammatory and antioxidant enzymes via the nuclear factor-erythroid 2-related factor 2 pathway to block tumor progression. A final highlight is the use of prodrugs activated by P450 enzymes to halt tumor growth and considerations of dosing schedule and targeted delivery of the P450 transgene to tumor tissue. In addition to highlighting recent successes in these areas, limitations and areas that should be targeted for further investigation are discussed.

Prospects for Clinical Development of Stat5 Inhibitor IST5-002: High Transcriptomic Specificity in Prostate Cancer and Low Toxicity In Vivo.

Stat5 is of significant interest in the search for new therapeutics for prostate cancer (PC) and hematopoietic disorders. We evaluated the transcriptomic specificity of the Stat5a/b inhibitor IST5-002 (IST5) in PC, defined more closely its mechanisms of action, and investigated the in vivo toxicity of IST5 for further optimization for clinical development. The transcriptomic specificity of IST5 vs. genetic Stat5 knockdown was evaluated by RNA-seq analysis, which showed high similarity with the Pearson correlation coefficient ranging from 0.98-0.99. The potency of IST5 vs. its derivative lacking the phosphate group in suppressing Stat5 was evaluated in two separate but complementary assays. The inhibitory activity of IST5 against kinases was investigated in cell-free assays followed by more focused evaluation in a cell-based assay. IST5 has no specific inhibitory activity against 54 kinases, while suppressing Stat5 phosphorylation and subsequent dimerization in PC cells. The phosphate group was not critical for the biological activity of IST5 in cells. The acute, sub-chronic and chronic toxicity studies of IST5 were carried out in mice. IST5 did not cause any significant toxic effects or changes in the blood profiles. The present work supports further optimization of IST5 for oral bioavailability for clinical development for therapies for solid tumors, hematological and myeloproliferative disorders.

Synergistic effect of a novel antiandrogen, VN/124-1, and signal transduction inhibitors in prostate cancer progression to hormone independence in vitro.

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.

17alpha-Hydroxylase/17,20 lyase inhibitor VN/124-1 inhibits growth of androgen-independent prostate cancer cells via induction of the endoplasmic reticulum stress response.

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.

Androgen receptor inactivation contributes to antitumor efficacy of 17{alpha}-hydroxylase/17,20-lyase inhibitor 3beta-hydroxy-17-(1H-benzimidazole-1-yl)androsta-5,16-diene in prostate cancer.

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.

The Novel Mnk1/2 Degrader and Apoptosis Inducer VNLG-152 Potently Inhibits TNBC Tumor Growth and Metastasis.

Currently, there are no effective therapies for patients with triple-negative breast cancer (TNBC), an aggressive and highly metastatic disease. Activation of eukaryotic initiation factor 4E (eIF4E) by mitogen-activated protein kinase (MAPK)-interacting kinases 1 and 2 (Mnk1/2) play a critical role in the development, progression and metastasis of TNBC. Herein, we undertook a comprehensive study to evaluate the activity of a first-in-class Mnk1/2 protein degraders, racemic VNLG-152R and its two enantiomers (VNLG-152E1 and VNLG-152E2) in in vitro and in vivo models of TNBC. These studies enabled us to identify racemic VNLG-152R as the most efficacious Mnk1/2 degrader, superior to its pure enantiomers. By targeting Mnk1/2 protein degradation (activity), VNLG-152R potently inhibited both Mnk-eIF4E and mTORC1 signaling pathways and strongly regulated downstream factors involved in cell cycle regulation, apoptosis, pro-inflammatory cytokines/chemokines secretion, epithelial-mesenchymal transition (EMT) and metastasis. Most importantly, orally bioavailable VNLG-152R exhibited remarkable antitumor (91 to 100% growth inhibition) and antimetastatic (~80% inhibition) activities against cell line and patient-derived TNBC xenograft models, with no apparent host toxicity. Collectively, these studies demonstrate that targeting Mnk-eIF4E/mTORC1 signaling with a potent Mnk1/2 degrader, VNLG-152R, is a novel therapeutic strategy that can be developed as monotherapy for the effective treatment of patients with primary/metastatic TNBC.

Galeterone and The Next Generation Galeterone Analogs, VNPP414 and VNPP433-3ß Exert Potent Therapeutic Effects in Castration-/Drug-Resistant Prostate Cancer Preclinical Models In Vitro and In Vivo.

These studies compared the efficacies of our clinical agent galeterone (Gal) and the FDA-approved prostate cancer drug, enzalutamide (ENZ) with two lead next generation galeterone analogs (NGGAs), VNPP414 and VNPP433-3ß, using prostate cancer (PC) in vitro and in vivo models. Antitumor activities of orally administered agents were also assessed in CWR22Rv1 tumor-bearing mice. We demonstrated that Gal and NGGAs degraded AR/AR-V7 and Mnk1/2; blocked cell cycle progression and proliferation of human PC cells; induced apoptosis; inhibited cell migration, invasion, and putative stem cell markers; and reversed the expression of epithelial-to-mesenchymal transition (EMT). In addition, Gal/NGGAs (alone or in combination) also inhibited the growth of ENZ-, docetaxel-, and mitoxantrone-resistant human PC cell lines. The NGGAs exhibited improved pharmacokinetic profiles over Gal in mice. Importantly, in vivo testing showed that VNPP433-3ß (at 7.53-fold lower equimolar dose than Gal) markedly suppressed (84% vs. Gal, 47%; p < 0.01) the growth of castration-resistant PC (CRPC) CWR22Rv1 xenograft tumors, with no apparent host toxicity. ENZ was ineffective in this CRPC xenograft model. In summary, our findings show that targeting AR/AR-V7 and Mnk1/2 for degradation represents an effective therapeutic strategy for PC/CRPC treatment and supports further development of VNPP433-3ß towards clinical investigation.