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.

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.

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.