Puranik Purushottamachar

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.

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 VNPP433-3β, 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 VNPP433-3β (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 VNPP433-3β towards clinical investigation.

A fact that is largely unknown in the lay press and even the scientific community is that today cancer kills more people worldwide than tuberculosis (TB), malaria, and human immunodeficiency virus (HIV) combined. Benzimidazole is a heterocyclic aromatic organic compound considered to be a useful pharmacophore in a variety of impactful drugs. The purpose of this review is to highlight the benzimidazole-containing agents that are currently in clinical use or in clinical development as anticancer drugs. It is hoped that this review would function as comprehensive working reference of research accomplishment in the field of discovery and development of benzimidazole-based anticancer drugs.

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.

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, in clinically relevant models of TNBC. These studies enabled us to identify racemic VNLG-152R as the most efficacious Mnk1/2 degrader. 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 and antimetastatic 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 effective treatment of patients with primary/metastatic TNBC.

Purpose Novel retinoic acid metabolism blocking agent (RAMBA), VN/12-1, is a highly potent anti-cancer agent that induces autophagy. Its combination with autophagy inhibitor chloroquine (CHL) has been shown to synergistically enhance apoptosis in breast cancer cells. The purpose of this study was to determine the toxicity and pharmacokinetic profile of VN/12-1 and its combination with CHL. Methods Preliminary toxicology of VN/12-1 was determined using female SCID mice (n = 4 for each group). ATRA was used for comparison. We selected four different doses of VN/12-1 and ATRA. Two of the doses were low and less frequent (2.5 and 5 mg/kg twice a week), and the remaining doses were high and more frequent (10 and 20 mg/kg every day). The dose of CHL was 50 mg/kg twice a week. For pharmacokinetic (PK) study, 20 mg/kg of VN/12-1 was injected subcutaneously (s.c.) into the mice, and their plasma was collected at various intervals (n = 2) and analyzed by HPLC. Results The lower and less frequent doses of VN/12-1 and ATRA were found to be least toxic. However, high and more frequent doses of these compounds were toxic to the mice. PK results showed that VN/12-1 has a half-life of 6 h. The area under the curve (AUC) for VN/12-1 was 83.78 h μg/ml. Conclusions VN/12-1 and ATRA are non-toxic when used as 5 mg/kg twice a week as single agents or in combination with CHL. The favorable PK properties of VN/12-1 can potentially be used for its further advanced pre-clinical and clinical development.