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 VNPP433-3β, 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.

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 VNPP433-3β which is potently efficacious against pre-clinical models of PCa. This study describes the mechanism of action of VNPP433-3β 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. VNPP433-3β 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, VNPP433-3β 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, VNPP433-3β 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.

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.

Cholesterol (CHOL) homeostasis is significantly modulated in prostate cancer (PCa) suggesting an active role in PCa development and progression. Several studies indicate a strong correlation between elevated CHOL levels and increased PCa risk and severity. Inhibition of CHOL biosynthesis at different steps, including lanosterol synthase (LSS), has shown significant efficacy against both hormone‐dependent and castration‐resistant PCa. Earlier, we reported proteasomal degradation of androgen receptor (AR)/AR‐Vs and Mnk1/2 as the primary mechanisms of action of VNPP433‐3β in inhibiting PCa cell proliferation and tumor growth. Through thermal proteome profiling, comparative proteomics and cellular thermal shift assay, we identified VNPP433‐3β's ancillary effect of lowering CHOL by binding to LSS and lanosterol 14‐alpha demethylase, potentially inhibiting CHOL biosynthesis in PCa cells and tumors. Additionally, in conjunction with our previously reported transcriptome analysis, proteomics reveals that VNPP433‐3β modulated upstream regulators and pathways critical for PCa stem cell maintenance and recurrence. The inhibition of CHOL biosynthesis by VNPP433‐3β reinforces its multifaceted effects in PCa across all stages, highlighting its potential as a single‐agent therapy. Achieving reduced CHOL levels aligns with better treatment outcomes, further substantiating VNPP433‐3β's therapeutic potential.