Introduction Metastatic castration-resistant prostate cancer (mCRPC) remains the second leading c... more Introduction Metastatic castration-resistant prostate cancer (mCRPC) remains the second leading cause of cancer-related death in men in the United States and the second most frequently diagnosed cancer among males worldwide. Dysregulated androgen receptor (AR) is associated with nearly all known cases of prostate cancer (PCa) and its expression level has been correlated with poor survival. This study describes the mechanism of action of VNPP433-3β, a next generation galeterone analog (NGGA) in inhibiting PCa. Methods PCa cell lines CWR22Rv1 and LNCaP were treated with VNPP433-3β and followed up using cellular thermal shift assay (CETSA), RNA seq, mRNA 5’ cap-binding assay, translation and proteasome inhibition assays. Co-immunoprecipitation, molecular docking and fluorescence spectroscopy were used to study the interactions. Results VNPP433-3β, the lead next generation galeterone analog (NGGA) directly interacts with AR and inhibits metastatic castration resistant PCa (mCRPC) by enh...
Targeted protein degradation is a fast-evolving therapeutic strategy to target even the tradition... more 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...
Prostate cancer (PCa) relies in part on AR-signaling for disease development and progression. Ear... more 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 consequen...
Currently, there are no effective therapies for patients with triple-negative breast cancer (TNBC... more 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 anti...
VN/12-1 is a novel retinoic acidmetabolism blocking agent discovered in our laboratory. The purpo... more VN/12-1 is a novel retinoic acidmetabolism blocking agent discovered in our laboratory. The purpose of the studywas to elucidate themolecularmechanismof anticancer activity ofVN/12-1 inbreast cancer cell lines and in tumor xenografts.We investigated the effects of VN/12-1 on induction of autophagy and apoptosis in SKBR3 cells. Furthermore, we also examined the impact of pharmacologic and genomic inhibition of autophagy on anticancer activity of VN/12-1. Finally, the antitumor activity of VN/12-1 was evaluated as a single agent and in combinationwith autophagy inhibitor chloroquine in an SKBR-3mouse xenograft model. Short exposure of low dose (<10 mmol/L) of VN/12-1 induced endoplasmic reticulum stress, autophagy, and inhibited G1–S phase transition and caused a protective response. However, a higher dose of VN/12-1 initiated apoptosis in vitro. Inhibition of autophagy using either pharmacologic inhibitors or RNA interference of Beclin-1 enhanced anticancer activity induced by VN/...
Stat5 is of significant interest in the search for new therapeutics for prostate cancer (PC) and ... more 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 phosphat...
These studies compared the efficacies of our clinical agent galeterone (Gal) and the FDA-approved... more 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 s...
Currently, there are no effective therapies for patients with triple-negative breast cancer (TNBC... more 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 t...
VNLG-152 is a novel retinamide (NR) shown to suppress growth and progression of genetically diver... more VNLG-152 is a novel retinamide (NR) shown to suppress growth and progression of genetically diverse prostate cancer cells via inhibition of androgen receptor signaling and eukaryotic initiation factor 4E (eIF4E) translational machinery. Herein, we report therapeutic effects of VNLG-152 on castration-resistant prostate cancer (CRPC) growth and metastatic phenotype in a CRPC tumor xenograft model. Administration of VNLG-152 significantly and dose-dependently suppressed the growth of aggressive CWR22Rv1 tumors by 63.4% and 76.3% at 10 and 20 mg·kg bw, respectively (P < 0.0001), vs. vehicle with no host toxicity. Strikingly, the expression of full-length androgen receptor (f-AR)/androgen receptor splice variant-7 (AR-V7), mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1/2), phosphorylated eIF4E and their associated target proteins, including prostate-specific antigen, cyclin D1 and Bcl-2, were strongly decreased in VNLG-152-treated tumors signifying inhibition of f-...
Introduction Metastatic castration-resistant prostate cancer (mCRPC) remains the second leading c... more Introduction Metastatic castration-resistant prostate cancer (mCRPC) remains the second leading cause of cancer-related death in men in the United States and the second most frequently diagnosed cancer among males worldwide. Dysregulated androgen receptor (AR) is associated with nearly all known cases of prostate cancer (PCa) and its expression level has been correlated with poor survival. This study describes the mechanism of action of VNPP433-3β, a next generation galeterone analog (NGGA) in inhibiting PCa. Methods PCa cell lines CWR22Rv1 and LNCaP were treated with VNPP433-3β and followed up using cellular thermal shift assay (CETSA), RNA seq, mRNA 5’ cap-binding assay, translation and proteasome inhibition assays. Co-immunoprecipitation, molecular docking and fluorescence spectroscopy were used to study the interactions. Results VNPP433-3β, the lead next generation galeterone analog (NGGA) directly interacts with AR and inhibits metastatic castration resistant PCa (mCRPC) by enh...
Targeted protein degradation is a fast-evolving therapeutic strategy to target even the tradition... more 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...
Prostate cancer (PCa) relies in part on AR-signaling for disease development and progression. Ear... more 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 consequen...
Currently, there are no effective therapies for patients with triple-negative breast cancer (TNBC... more 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 anti...
VN/12-1 is a novel retinoic acidmetabolism blocking agent discovered in our laboratory. The purpo... more VN/12-1 is a novel retinoic acidmetabolism blocking agent discovered in our laboratory. The purpose of the studywas to elucidate themolecularmechanismof anticancer activity ofVN/12-1 inbreast cancer cell lines and in tumor xenografts.We investigated the effects of VN/12-1 on induction of autophagy and apoptosis in SKBR3 cells. Furthermore, we also examined the impact of pharmacologic and genomic inhibition of autophagy on anticancer activity of VN/12-1. Finally, the antitumor activity of VN/12-1 was evaluated as a single agent and in combinationwith autophagy inhibitor chloroquine in an SKBR-3mouse xenograft model. Short exposure of low dose (<10 mmol/L) of VN/12-1 induced endoplasmic reticulum stress, autophagy, and inhibited G1–S phase transition and caused a protective response. However, a higher dose of VN/12-1 initiated apoptosis in vitro. Inhibition of autophagy using either pharmacologic inhibitors or RNA interference of Beclin-1 enhanced anticancer activity induced by VN/...
Stat5 is of significant interest in the search for new therapeutics for prostate cancer (PC) and ... more 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 phosphat...
These studies compared the efficacies of our clinical agent galeterone (Gal) and the FDA-approved... more 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 s...
Currently, there are no effective therapies for patients with triple-negative breast cancer (TNBC... more 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 t...
VNLG-152 is a novel retinamide (NR) shown to suppress growth and progression of genetically diver... more VNLG-152 is a novel retinamide (NR) shown to suppress growth and progression of genetically diverse prostate cancer cells via inhibition of androgen receptor signaling and eukaryotic initiation factor 4E (eIF4E) translational machinery. Herein, we report therapeutic effects of VNLG-152 on castration-resistant prostate cancer (CRPC) growth and metastatic phenotype in a CRPC tumor xenograft model. Administration of VNLG-152 significantly and dose-dependently suppressed the growth of aggressive CWR22Rv1 tumors by 63.4% and 76.3% at 10 and 20 mg·kg bw, respectively (P < 0.0001), vs. vehicle with no host toxicity. Strikingly, the expression of full-length androgen receptor (f-AR)/androgen receptor splice variant-7 (AR-V7), mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1/2), phosphorylated eIF4E and their associated target proteins, including prostate-specific antigen, cyclin D1 and Bcl-2, were strongly decreased in VNLG-152-treated tumors signifying inhibition of f-...
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