Andrew K. Kwegyir-Afful

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.

Galeterone and VNPT55 disrupt Mnk‐eIF4E to inhibit prostate cancer cell migration and invasion

Metastatic castration‐resistant prostate cancer (mCRPC) accounts for a high percentage of prostate cancer mortality. The proprietary compound galeterone (gal) was designed to inhibit proliferation of androgen/androgen receptor (AR)‐dependent prostate cancer cell in vitro and in vivo and is currently in phase III clinical development. Additionally, clinical studies with gal revealed its superb efficacy in four different cohorts of patients with mCRPC, including those expressing splice variant AR‐V7. Preclinical studies with gal show that it also exhibits strong antiproliferative activities against AR‐negative prostate cancer cells and tumors through a mechanism involving phosphorylation of eIF2α, which forms an integral component of the eukaryotic mRNA translation complex. Thus, we hypothesized that gal and its new analog, VNPT55, could modulate oncogenic mRNA translation and prostate cancer cell migration and invasion. We report that gal and VNPT55 profoundly inhibit migration and invasion of prostate cancer cells, possibly by down‐regulating protein expression of several EMT markers (Snail, Slug, N‐cadherin, vimentin, and MMP‐2/‐9) via antagonizing the Mnk–eIF4E axis. In addition, gal/VNPT55 inhibited both NF‐κB and Twist1 transcriptional activities, down‐regulating Snail and BMI‐1 mRNA expression, respectively. Furthermore, profound up‐regulation of E‐cadherin mRNA and protein expression may explain the observed significant inhibition of prostate cancer cell migration and invasion. Moreover, expression of self‐renewal proteins, β‐catenin, CD44, and Nanog, was markedly depleted. Analysis of gal/VNPT55‐treated CWR22Rv1 xenograft tissue sections also revealed that observations in vitro were recapitulated in vivo. Our results suggest that gal/VNPT55 could become promising agents for the prevention and/or treatment of all stages of prostate cancer.

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.

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

Galeterone and its analogs inhibit Mnk-eIF4E axis, synergize with gemcitabine, impede pancreatic cancer cell migration, invasion and proliferation and inhibit tumor growth in mice

Survival rate for pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is poor, with about 80% of patients presenting with the metastatic disease. Gemcitabine, the standard chemotherapeutic agent for locally advanced and metastatic PDAC has limited efficacy, attributed to innate/acquired resistance and activation of pro-survival pathways. The Mnk1/2-eIF4E and NF-κB signaling pathways are implicated in PDAC disease progression/metastasis and also associated with gemcitabine-induced resistance in PDAC. Galeterone (gal), a multi-target, agent in phase III clinical development for prostate cancer has also shown effects on the aforementioned pathways. We show for the first time, that gal/analogs (VNPT55, VNPP414 and VNPP433-3β) profoundly inhibited cell viability of gemcitabine-naive/resistance PDAC cell lines and strongly synergized with gemcitabine in gemcitabine-resistant PDAC cells. In addition, to inducing G1 cell cycle arrest, gal/analogs induced caspase 3-mediated cell-death of PDAC cells. Gal/analogs caused profound downregulation of Mnk1/2, peIF4E and NF-κB (p-p65), metastatic inducing factors (N-cadherin, MMP-1/-2/-9, Slug, Snail and CXCR4) and putative stem cell factors, (β-Catenin, Nanog, BMI-1 and Oct-4). Gal/analog also depleted EZH2 and upregulated E-Cadherin. These effects resulted in significant inhibition of PDAC cell migration, invasion and proliferation. Importantly, we also observed strong MiaPaca-2 tumor xenograft growth inhibition (61% to 92%). Collectively, these promising findings strongly support further development of gal/analogs as novel therapeutics for PDAC.Survival rate for pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) is poor, with about 80% of patients presenting with the metastatic disease. Gemcitabine, the standard chemotherapeutic agent for locally advanced and metastatic PDAC has limited efficacy, attributed to innate/acquired resistance and activation of pro-survival pathways. The Mnk1/2-eIF4E and NF-κB signaling pathways are implicated in PDAC disease progression/metastasis and also associated with gemcitabine-induced resistance in PDAC. Galeterone (gal), a multi-target, agent in phase III clinical development for prostate cancer has also shown effects on the aforementioned pathways. We show for the first time, that gal/analogs (VNPT55, VNPP414 and VNPP433-3β) profoundly inhibited cell viability of gemcitabine-naive/resistance PDAC cell lines and strongly synergized with gemcitabine in gemcitabine-resistant PDAC cells. In addition, to inducing G1 cell cycle arrest, gal/analogs induced caspase 3-mediated cell-death of PDAC cells. Gal/analogs caused profound downregulation of Mnk1/2, peIF4E and NF-κB (p-p65), metastatic inducing factors (N-cadherin, MMP-1/-2/-9, Slug, Snail and CXCR4) and putative stem cell factors, (β-Catenin, Nanog, BMI-1 and Oct-4). Gal/analog also depleted EZH2 and upregulated E-Cadherin. These effects resulted in significant inhibition of PDAC cell migration, invasion and proliferation. Importantly, we also observed strong MiaPaca-2 tumor xenograft growth inhibition (61% to 92%). Collectively, these promising findings strongly support further development of gal/analogs as novel therapeutics for PDAC.

Abstract B173: Galeterone shows anti-tumor activity in multiple pre-clinical models that express androgen receptor splice variants, supporting correlative patient data seen in ARMOR2

Multiple experimental approaches were used to examine whether and by what mechanism galeterone affects prostate cancer growth in cells and tumors expressing androgen receptor (AR) splice variants, such as AR-V7. Galeterone is a selective, multitargeted, small molecule that disrupts androgen signaling at multiple points in the pathway. ARMOR3-SV is a Phase 3, randomized, open-label, multicenter, controlled clinical trial in metastatic castration resistant prostate cancer (mCRPC) patients whose tumors express AR splice variant-7 mRNA (AR-V7). AR-V7 is a truncated, constitutively active splice variant of the AR that lacks the ligand binding domain (LBD) and has been implicated in prostate cancer progression. The expression of AR-V7 occurs in approximately 14-26% of men with mCRPC prior to second-generation anti-androgens or chemotherapy, and AR-V7 expression has been clinically associated with resistance to enzalutamide (Xtandi) or abiraterone (Zytiga). Here we provide the pre-clinical and clinical rationale for the development of galeterone in mCRPC patients with AR-V7. Our findings show that using multiple pre-clinical approaches, galeterone reduces in vitro prostate cancer cell proliferation, androgen receptor signaling and xenograft tumor growth using cells and tumors that express AR splice variants. Reductions in AR splice variant protein were also observed in cells that also co-express wild-type AR, and prostate cancer cells that lack wild type AR, but transiently express AR splice variant protein. Clinical data further support activity of galeterone in patients with truncated androgen receptors. Galeterone9s unique mechanism of AR protein downregulation occurs in both splice variant and wild-type AR, supporting that galeterone-induced AR downregulation is independent of the AR LBD. Because preclinical and clinical data support an opportunity for treatment in this setting, Tokai Pharmaceuticals designed ARMOR3-SV to test galeterone vs. enzalutamide in men with metastatic castration-resistant prostate cancer whose tumors express the AR-V7 splice variant. Citation Format: Douglas B. Jacoby, Amina Zoubeidi, Eva B. Corey, Elahe Mostaghel, Andrew K. Kwegyir-Afful, Senthilmurugan Ramalingam, Vincent Njar. Galeterone shows anti-tumor activity in multiple pre-clinical models that express androgen receptor splice variants, supporting correlative patient data seen in ARMOR2. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B173.

Abstract 756: First MNK degrading agents block phosphorylation of eIF4E, induce apoptosis, and inhibit cell growth, migration and invasion in triple-negative and HER2-overexpressing breast cancer cell lines

Introduction: Retinoic acid metabolism blocking agents are known to exhibit a range of anticarcinogenic properties. This study investigated the anticancer efficacy of novel, potent retinamide retinoic acid metabolism blocking agents (RRs) in triple negative and Her 2 overexpressing breast cancer cells. Recent findings suggest that overexpression of eukaryotic translation initiation factor 4E (eIF4E) in breast cancers critically augments CAP-dependent mRNA translation and synthesis of proteins involved in cell growth, cell proliferation, invasion and apoptosis evasion. The oncogenic potential of eIF4E is strictly dependent on serine 209 phosphorylation by upstream MAPK-interacting kinases (Mnk). Targeting Mnk/eIF4E pathway for blocking Mnk function and eIF4E phosphorylation is therefore a novel approach for treating breast cancers particularly for HER-2-positive and triple negative breast cancers that have no indications for endocrine therapy or effective treatment regimes. Methods: Triple negative and Her 2 overexprssing breast cancer cells were treated with retinamide RAMBAs and Mnk inhibitors and evaluated the effects of RRs on growth inhibition, MAPK/Mnk mediated eIF4E protein translational machinery and downstream biological effects in triple negative and Her 2 overexpressing breast cancer cells Results: We report that the degradation of Mnk by RRs in breast cancer cells blocks eIF4E phosphorylation and subsequently inhibits cell growth, invasion and metastasis in addition to inducing apoptosis. Most importantly the anticancer efficacy of RRs was mediated via degrading Mnk rather than inhibiting its kinase activity like Mnk inhibitors (Cercosporamide and CGP 57380). RRs effect on p-eIF4E downregulation and growth inhibition were also far more potent than the clinically relevant retinoids and Mnk inhibitors, cercosporamide and CGP 57380. Conclusion: Together our findings provide the first preclinical proof-of-concept of novel Mnk degrading agents for Mnk based therapeutic treatment of breast cancers. Citation Format: Senthilmurugan Ramalingam, Lalji Gediya, Puranik Purushottamachar, Andrew Kwegyir-Afful, Vidya priyadarsini Ramamurthy, Hannah Mbatia, Vincent Njar. First MNK degrading agents block phosphorylation of eIF4E, induce apoptosis, and inhibit cell growth, migration and invasion in triple-negative and HER2-overexpressing breast cancer cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 756. doi:10.1158/1538-7445.AM2014-756