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.

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.

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.

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.

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.

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.

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.

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.

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.

Novel galeterone analogs act independently of AR and AR-V7 for the activation of the unfolded protein response and induction of apoptosis in the CWR22Rv1 prostate cancer cell model.

The androgen receptor (AR) has long been the primary target for the treatment of prostate cancer (PC). Despite continuous efforts to block AR activity through ligand depletion, AR antagonism, AR depletion and combinations thereof, advanced PC tumors remain resilient. Herein, we evaluate two galeterone analogs, VNPT-178 and VNLG-74A, in PC cell models of diverse androgen and AR dependence attempting to delineate their mechanisms of action and potential clinical utility. Employing basic biochemical techniques, we determined that both analogs have improved antiproliferative and anti-AR activities compared to FDA-approved abiraterone and enzalutamide. However, induction of apoptosis in these models is independent of the AR and its truncated variant, AR-V7, and instead likely results from sustained endoplasmic reticulum stress and deregulated calcium homeostasis. Using in silico molecular docking, we predict VNPT-178 and VNLG-74A bind the ATPase domain of BiP/Grp78 and Hsp70-1A with greater affinity than the AR. Disruption of 70 kDa heat shock protein function may be the underlying mechanism of action for these galeterone analogs. Therefore, despite simultaneously antagonizing AR activity, AR and/or AR-V7 expression alone may inadequately predict a patient's response to treatment with VNPT-178 or VNLG-74A. Future studies evaluating the context-specific limitations of these compounds may provide clarity for their clinical application.

Galeterone and its analogs inhibit Mnk-eIF4E axis, synergize with gemcitabine, impede pancreatic cancer cell migration, invasion and proliferation and inhibit tumor growth in mice.

Survival rate for pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is poor, with about 80% of patients presenting with the metastatic disease. Gemcitabine, the standard chemotherapeutic agent for locally advanced and metastatic PDAC has limited efficacy, attributed to innate/acquired resistance and activation of pro-survival pathways. The Mnk1/2-eIF4E and NF-κB signaling pathways are implicated in PDAC disease progression/metastasis and also associated with gemcitabine-induced resistance in PDAC. Galeterone (gal), a multi-target, agent in phase III clinical development for prostate cancer has also shown effects on the aforementioned pathways. We show for the first time, that gal/analogs (VNPT55, VNPP414 and VNPP433-3ß) profoundly inhibited cell viability of gemcitabine-naive/resistance PDAC cell lines and strongly synergized with gemcitabine in gemcitabine-resistant PDAC cells. In addition, to inducing G1 cell cycle arrest, gal/analogs induced caspase 3-mediated cell-death of PDAC cells. Gal/analogs caused profound downregulation of Mnk1/2, peIF4E and NF-κB (p-p65), metastatic inducing factors (N-cadherin, MMP-1/-2/-9, Slug, Snail and CXCR4) and putative stem cell factors, (ß-Catenin, Nanog, BMI-1 and Oct-4). Gal/analog also depleted EZH2 and upregulated E-Cadherin. These effects resulted in significant inhibition of PDAC cell migration, invasion and proliferation. Importantly, we also observed strong MiaPaca-2 tumor xenograft growth inhibition (61% to 92%). Collectively, these promising findings strongly support further development of gal/analogs as novel therapeutics for PDAC.

Galeterone and VNPT55 disrupt Mnk-eIF4E to inhibit prostate cancer cell migration and invasion.

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, ß-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.

Identification of Novel Steroidal Androgen Receptor Degrading Agents Inspired by Galeterone 3ß-Imidazole Carbamate.

Degradation of all forms of androgen receptors (ARs) is emerging as an advantageous therapeutic paradigm for the effective treatment of prostate cancer. In continuation of our program to identify and develop improved efficacious novel small-molecule agents designed to disrupt AR signaling through enhanced AR degradation, we have designed, synthesized, and evaluated novel C-3 modified analogues of our phase 3 clinical agent, galeterone (5). Concerns of potential in vivo stability of our recently discovered more efficacious galeterone 3ß-imidazole carbamate (6) led to the design and synthesis of new steroidal compounds. Two of the 11 compounds, 3ß-pyridyl ether (8) and 3ß-imidazole (17) with antiproliferative GI50 values of 3.24 and 2.54 µM against CWR22Rv1 prostate cancer cell, are 2.75- and 3.5-fold superior to 5. In addition, compounds 8 and 17 possess improved (∼4-fold) AR-V7 degrading activities. Importantly, these two compounds are expected to be metabolically stable, making them suitable for further development as new therapeutics against all forms of prostate cancer.

Galeterone and VNPT55 induce proteasomal degradation of AR/AR-V7, induce significant apoptosis via cytochrome c release and suppress growth of castration resistant prostate cancer xenografts in vivo.

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.

Structure-Based Screen Identifies a Potent Small Molecule Inhibitor of Stat5a/b with Therapeutic Potential for Prostate Cancer and Chronic Myeloid Leukemia.

Bypassing tyrosine kinases responsible for Stat5a/b phosphorylation would be advantageous for therapy development for Stat5a/b-regulated cancers. Here, we sought to identify small molecule inhibitors of Stat5a/b for lead optimization and therapy development for prostate cancer and Bcr-Abl-driven leukemias. In silico screening of chemical structure databases combined with medicinal chemistry was used for identification of a panel of small molecule inhibitors to block SH2 domain-mediated docking of Stat5a/b to the receptor-kinase complex and subsequent phosphorylation and dimerization. We tested the efficacy of the lead compound IST5-002 in experimental models and patient samples of two known Stat5a/b-driven cancers, prostate cancer and chronic myeloid leukemia (CML). The lead compound inhibitor of Stat5-002 (IST5-002) prevented both Jak2 and Bcr-Abl-mediated phosphorylation and dimerization of Stat5a/b, and selectively inhibited transcriptional activity of Stat5a (IC50 = 1.5µmol/L) and Stat5b (IC50 = 3.5 µmol/L). IST5-002 suppressed nuclear translocation of Stat5a/b, binding to DNA and Stat5a/b target gene expression. IST5-002 induced extensive apoptosis of prostate cancer cells, impaired growth of prostate cancer xenograft tumors, and induced cell death in patient-derived prostate cancers when tested ex vivo in explant organ cultures. Importantly, IST5-002 induced robust apoptotic death not only of imatinib-sensitive but also of imatinib-resistant CML cell lines and primary CML cells from patients. IST5-002 provides a lead structure for further chemical modifications for clinical development for Stat5a/b-driven solid tumors and hematologic malignancies.

VN/14-1 induces ER stress and autophagy in HP-LTLC human breast cancer cells and has excellent oral pharmacokinetic profile in female Sprague Dawley rats.

Resistance to aromatase inhibitors is a major concern in the treatment of breast cancer. Long-term letrozole cultured (LTLC) cells represent a model of resistance to aromatase inhibitors. The LTLC cells were earlier generated by culturing MCF-7Ca, the MCF-7 human breast cancer cell line stably transfected with human placental aromatase gene for a prolonged period in the presence of letrozole. In the present study the effect of RAMBA, VN/14-1 on the sensitivity of LTLC cells upon multiple passaging and the mechanisms of action of VN/14-1 in such high passage LTLC (HP-LTLC) cells was investigated. We report that multiple passaging of LTLC cells (HP-LTLC cell clones) led to profound decrease in their sensitivity to VN/14-1. Additionally, microarray studies and protein analysis revealed that VN/14-1 induced marked endoplasmic reticulum (ER) stress and autophagy in HP-LTLC cells. We further report that VN/14-1 in combination with thapsigargin exhibited synergistic anti-cancer effect in HP-LTLC cells. Preliminary pharmacokinetics in rats revealed that VN/14-1 reached a peak plasma concentration (Cmax) within 0.17h after oral dosing. Its absolute oral bioavailability was >100%. Overall these results indicate potential of VN/14-1 for further clinical development as a potential oral agent for the treatment of breast cancer.

First MNKs degrading agents block phosphorylation of eIF4E, induce apoptosis, inhibit cell growth, migration and invasion in triple negative and Her2-overexpressing breast cancer cell lines.

Some retinoic acid metabolism blocking agents (RAMBAs) are known to exhibit a wide range of anticancer activities by mechanisms that are still not completely resolved. This study investigated the anticancer efficacy and mechanism(s) of novel RAMBA retinamides (RRs) in triple negative and Her-2 overexpressing breast cancer cells. Specifically, we examined the possibility that RRs affect the translational machinery in these breast cancer (BC) cells. Recent findings suggest that overexpression of eukaryotic translation initiation factor 4E (eIF4E) in breast cancers critically augments CAP-dependent mRNA translation and synthesis of proteins involved in cell growth, cell proliferation, invasion and apoptosis evasion. The oncogenic potential of eIF4E is strictly dependent on serine209 phosphorylation by upstream MAPK-interacting kinases (Mnks). Targeting Mnk/eIF4E pathway for blocking Mnk function and eIF4E phosphorylation is therefore a novel approach for treating BCs, particularly for Her2-positive and triple negative breast cancers that have no indications for endocrine therapy or effective treatment regimes. We report for the first time that the degradation of Mnk1 by RRs in BC cells blocks eIF4E phosphorylation and subsequently inhibits cell growth, colonization, invasion, and migration and induce apoptosis. Most importantly, the anticancer efficacy of RRs was mediated via degrading Mnk rather than inhibiting its kinase activity like Mnk inhibitors (cercosporamide and CGP57380). Furthermore, RRs potencies on peIF4E down-regulation and growth inhibition were superior to those of two clinically relevant retinoids and the Mnk inhibitors. Together our findings provide the first preclinical proof-of-concept of novel Mnk degrading agents for Mnk/eIF4E based therapeutic treatment of breast cancers.

Systematic structure modifications of multitarget prostate cancer drug candidate galeterone to produce novel androgen receptor down-regulating agents as an approach to treatment of advanced prostate cancer.

As part of our program to explore the influence of small structural modifications of our drug candidate 3ß-(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 3ß-(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 3ß-(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.

A new simple and high-yield synthesis of 5α-dihydrotestosterone (DHT), a potent androgen receptor agonist.

We have devised an efficient procedure for the synthesis of 5α-dihydrotestosterone (DHT) (1) starting from 3ß-hydroxy-5α-androstan-17-one, providing the product in unprecedented 82% yield. A reported method of using toxic Jones reagent is replaced by milder oxidizing agent (NMO/TPAP) in the synthesis of a key intermediate 17ß-[(tert-butyldimethylsilyl)oxy]-5α-androstan-3-one (18). This new procedure is simple, does not require special apparatus/precautions or chromatographic purification in most of the steps.