Senthilmurugan Ramalingam

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 3β-(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 3β-(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 3β-(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.

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.

Novel C-4 Heteroaryl 13-cis-Retinamide Mnk/AR Degrading Agents Inhibit Cell Proliferation and Migration and Induce Apoptosis in Human Breast and Prostate Cancer Cells and Suppress Growth of MDA-MB-231 Human Breast and CWR22Rv1 Human Prostate Tumor Xenografts in Mice

The synthesis and in vitro and in vivo antibreast and antiprostate cancers activities of novel C-4 heteroaryl 13-cis-retinamides that modulate Mnk-eIF4E and AR signaling are discussed. Modifications of the C-4 heteroaryl substituents reveal that the 1H-imidazole is essential for high anticancer activity. The most potent compounds against a variety of human breast and prostate cancer (BC/PC) cell lines were compounds 16 (VNHM-1-66), 20 (VNHM-1-81), and 22 (VNHM-1-73). In these cell lines, the compounds induce Mnk1/2 degradation to substantially suppress eIF4E phosphorylation. In PC cells, the compounds induce degradation of both full-length androgen receptor (fAR) and splice variant AR (AR-V7) to inhibit AR transcriptional activity. More importantly, VNHM-1-81 has strong in vivo antibreast and antiprostate cancer activities, while VNHM-1-73 exhibited strong in vivo antibreast cancer activity, with no apparent host toxicity. Clearly, these lead compounds are strong candidates for development for the treatments of human breast and prostate cancers.

Dissecting major signaling pathways in prostate cancer development and progression: Mechanisms and novel therapeutic targets.

Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous malignancy and leading cause of cancer mortality in men. At the initial stages, prostate cancer is dependent upon androgens for their growth and hence effectively combated by androgen deprivation therapy (ADT). However, most patients eventually recur with an androgen deprivation-resistant phenotype, referred to as castration-resistant prostate cancer (CRPC), a more aggressive form for which there is no effective therapy presently available. The current review is an attempt to cover and establish an understanding of some major signaling pathways implicated in prostate cancer development and castration-resistance, besides addressing therapeutic strategies that targets the key signaling mechanisms.

Targeting of protein translation as a new treatment paradigm for prostate cancer.

Purpose of review The current overview will summarize some of the developments in the area of protein translation, including their relation to the therapeutic targeting of prostate cancer. Recent findings Translational control, mediated by the rate-limiting eukaryotic translation initiation factor 4E (eIF4E), drives selective translation of several oncogenic proteins, thereby contributing to tumor growth, metastasis, and treatment resistance in various cancers, including prostate cancer. As an essential regulatory hub, several oncogenic hyperactive signaling pathways appear to converge on eIF4E to promote tumorigenesis. Several approaches that target the eIF4E-dependent protein translation network are being actively studied, and it is likely that some may ultimately emerge as promising anticancer therapeutics. Summary An array of inhibitors has shown promise in targeting specific components of the translational machinery in several preclinical models of prostate cancer. It is hoped that some of these approaches may ultimately have relevance in improving the clinical outcomes of patients with advanced prostate cancer.

Identification of Novel Steroidal Androgen Receptor Degrading Agents Inspired by Galeterone 3β-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 3β-imidazole carbamate (6) led to the design and synthesis of new steroidal compounds. Two of the 11 compounds, 3β-pyridyl ether (8) and 3β-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.

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.

Abstract 1322: Clinical candidate galeterone (VN/124-1 or TOK-001) induces the degradation of full-length and splice variant androgen receptors in human prostate cancer cell lines via PI3K-Akt-Mdm2 pathway: implications for prostate cancer therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Galeterone (VN/124-1; TOK-001; 3β-hydroxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene) is a proprietary oral small molecule under clinical development for the treatment of castration resistant prostate cancer (CRPC). Galeterone (hereafter referred to as gal) disrupts androgen receptor (AR) signaling through a unique triple mechanism of action, including inhibition of CYP17 (androgen synthesis inhibition), AR antagonism and degradation of AR. Although we have previously unraveled the mechanisms of CYP17 inhibition and AR antagonism, the mechanism of AR degradation is still being elucidated. Here, we report for the first time the mechanism of gal -induced AR degradation, interestingly, gal also depletes splice variant AR in human prostate cancer cells. We evaluated the effects of gal on full-length (wild-type and mutant) and truncated (splice variant) AR in LNCaP, LAPC4 and CWR22Rv1 cells. Real-time PCR analysis of AR mRNA levels led to the hypothesis that gals effect on AR occurs through a post-translational mechanism(s). The 26S proteasome inhibitor (MG132) blocked the effect of gal on AR depletion, thus suggesting that gal induces AR degradation via the ubiquitin-proteasome pathway. In addition, gal caused increased phosphorylation of Akt and Mdm2, and pretreatment with the PI3K inhibitor, wortmannin, robustly inhibited gal-dependent AR depletion. Targeted knockdown of Mdm2 with siRNA also inhibited the depletion of AR. Antibody pull-down data also indicated that gal treatment enhanced the formation of complex between Akt, Mdm2 and AR which promote phosphorylation-dependent AR ubiquitination and its degradation by proteasome. The possible involvement of the proteasome-independent pathway (caspase-dependent) was eliminated because AR protein was reduced by gal to similar extents in the presence or absence of the pan caspase inhibitor, z-vad-fmk. Thus, in prostate cancer cells, our data support a model where gal induces phosphorylation of AR and Mdm2 through the PI3K/Akt pathway. AR then undergoes ubiquitination by Mdm2 E3 ligase followed by AR degradation by proteasome. Using Western blot analyses and MTT cell viability assays, gal exhibited strong correlation between its ability to deplete AR and reduce cell viability. We also show for the first time that gal and some new analogs can degrade both full-length and truncated AR3 in CWR22Rv1. In conclusion, because gal is currently being evaluated for clinical use against CRPC, its mechanism of AR degradation is of considerable clinical relevance. These discoveries of gal should generate excitement because it appears to be the only agent currently in clinical trial targeting CRPC progression driven by both full-length and splice variants AR. Citation Format: Andrew K. kwegyir-Afful, Senthilmurugan Ramalingam, Puranik Purushottamachar, Vincent C. O. Njar. Clinical candidate galeterone (VN/124-1 or TOK-001) induces the degradation of full-length and splice variant androgen receptors in human prostate cancer cell lines via PI3K-Akt-Mdm2 pathway: implications for prostate cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1322. doi:10.1158/1538-7445.AM2013-1322

The Novel Mnk1/2 Degrader 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, 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.

The retinamide VNLG‐152 inhibits f‐AR/AR‐V7 and MNK–eIF4E signaling pathways to suppress EMT and castration‐resistant prostate cancer xenograft growth

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−1 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‐AR/AR‐V7 and MNK–eIF4E signaling in VNLG‐152‐treated CWR22Rv1 tumors as observed in vitro. VNLG‐152 also suppressed the epithelial to mesenchymal transition in CWR22Rv1 tumors as evidenced by repression of N‐cadherin, β‐catenin, claudin, Slug, Snail, Twist, vimentin and matrix metalloproteinases (MMP‐2 and MMP‐9) with upsurge in E‐cadherin. These results highlight the promising use of VNLG‐152 in CRPC therapy and justify its further development towards clinical trials.