Carmen Adriana Banuelos

Regression of Castrate-Recurrent Prostate Cancer by a Small-Molecule Inhibitor of the Amino-Terminus Domain of the Androgen Receptor

Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.

An androgen receptor N-terminal domain antagonist for treating prostate cancer

Hormone therapies for advanced prostate cancer target the androgen receptor (AR) ligand-binding domain (LBD), but these ultimately fail and the disease progresses to lethal castration-resistant prostate cancer (CRPC). The mechanisms that drive CRPC are incompletely understood, but may involve constitutively active AR splice variants that lack the LBD. The AR N-terminal domain (NTD) is essential for AR activity, but targeting this domain with small-molecule inhibitors is complicated by its intrinsic disorder. Here we investigated EPI-001, a small-molecule antagonist of AR NTD that inhibits protein-protein interactions necessary for AR transcriptional activity. We found that EPI analogs covalently bound the NTD to block transcriptional activity of AR and its splice variants and reduced the growth of CRPC xenografts. These findings suggest that the development of small-molecule inhibitors that bind covalently to intrinsically disordered proteins is a promising strategy for development of specific and effective anticancer agents.

Niphatenones, glycerol ethers from the sponge Niphates digitalis block androgen receptor transcriptional activity in prostate cancer cells: structure elucidation, synthesis, and biological activity.

Extracts of the marine sponge Niphates digitalis collected in Dominica showed strong activity in a cell-based assay designed to detect antagonists of the androgen receptor (AR) that could act as lead compounds for the development of a new class of drugs to treat castration recurrent prostate cancer (CRPC). Assay-guided fractionation showed that niphatenones A (3) and B (4), two new glycerol ether lipids, were the active components of the extracts. The structures of 3 and 4 were elucidated by analysis of NMR and MS data and confimed via total synthesis. Biological evaluation of synthetic analogues of the niphatenones has shown that the enantiomers 7 and 8 are more potent than the natural products in the screening assay and defined preliminary SAR for the new AR antagonist pharmacophore, including the finding that the Michael acceptor enone functionality is not required for activity. Niphatenone B (4) and its enantiomer 8 blocked androgen-induced proliferation of LNCaP prostate cancer cells but had no effect on the proliferation of PC3 prostate cancer cells that do not express functional AR, consistent with activity as AR antagonists. Use of the propargyl ether 44 and Click chemistry showed that niphatenone B binds covalently to the activation function-1 (AF1) region of the AR N-terminus domain (NTD).

Radiosensitization by the Histone Deacetylase Inhibitor PCI-24781

Purpose: PCI-24781 is a novel broad spectrum histone deacetylase inhibitor that is currently in phase I clinical trials. The ability of PCI-24781 to act as a radiation sensitizer and the mechanisms of radiosensitization were examined. Experimental Design: Exponentially growing human SiHa cervical and WiDr colon carcinoma cells were exposed to 0.1 to 10 μmol/L PCI-24781 in vitro for 2 to 20 h before irradiation and 0 to 4 h after irradiation. Single cells and sorted populations were analyzed for histone acetylation, H2AX phosphorylation, cell cycle distribution, apoptotic fraction, and clonogenic survival. Results: PCI-24781 treatment for 4 h increased histone H3 acetylation and produced a modest increase in γH2AX but negligible cell killing or radiosensitization. Treatment for 24 h resulted in up to 80% cell kill and depletion of cells in S phase. Toxicity reached maximum levels at a drug concentration of ∼1 μmol/L, and cells in G1 phase at the end of treatment were preferentially spared. A similar dose-modifying factor (DMF0.1 = 1.5) was observed for SiHa cells exposed for 24 h at 0.1 to 3 μmol/L, and more radioresistant WiDr cells showed less sensitization (DMF0.1 = 1.2). Limited radiosensitization and less killing were observed in noncycling human fibroblasts. Cell sorting experiments confirmed that depletion of S-phase cells was not a major mechanism of radiosensitization and that inner noncycling cells of SiHa spheroids could be sensitized by nontoxic doses. PCI-24781 pretreatment increased the fraction of cells with γH2AX foci 24 h after irradation but did not affect the initial rate of loss of radiation-induced γH2AX or the rate of rejoining of DNA double-strand breaks. Conclusions: PCI-24781 shows promise as a radiosensitizing agent that may compromise the accuracy of repair of radiation damage.

N-terminal targeting of androgen receptor variant enhances response of castration resistant prostate cancer to taxane chemotherapy

Taxane‐based chemotherapy is an effective treatment for castration‐resistant‐prostate cancer (CRPC) via stabilization of microtubules. Previous studies identified that the inhibitory effect of microtubule‐targeting chemotherapy on androgen receptor (AR) activity was conferred by interfering with AR intracellular trafficking. The N‐terminal domain (NTD) of AR was identified as a tubulin‐interacting domain that can be effectively targeted by the novel small molecule inhibitor, EPI. Taken together this evidence provided the rationale that targeting AR nuclear translocation and activity via a combination of an antagonist of the AR NTD and taxane‐based chemotherapy may enhance the therapeutic response in CRPC. The present study investigated the anti‐tumor efficacy of a combination of EPI with Docetaxel chemotherapy, in cell models of CRPC, harboring the AR splice variants in addition to the full length AR. Our findings demonstrate that there was no significant effect on the androgen‐mediated nuclear transport of AR variants and AR transcriptional activity by Docetaxel. The therapeutic response to Docetaxel was enhanced by inhibition of the NTD of AR (by EPI) through cycling of epithelial‐mesenchymal‐transition (EMT) to mesenchymal‐epithelial‐transition (MET) among prostate cancer epithelial cells. These results support that transient “programming” of EMT by the AR NTD inhibitor, potentially drives the sensitivity of prostate tumors with differential distribution of AR variants to microtubule‐targeting chemotherapy. This study is of major significance in dissecting mechanisms to overcome taxane resistance in advanced CRPC.

Targeting Androgen Receptor Activation Function-1 with EPI to Overcome Resistance Mechanisms in Castration-Resistant Prostate Cancer.

Purpose: Persistent androgen receptor (AR) transcriptional activity is clinically evident in castration-resistant prostate cancer (CRPC). Therefore, AR remains as a viable therapeutic target for CRPC. All current hormonal therapies target the C-terminus ligand-binding domain (LBD) of AR. By using EPI to target AR activation function-1 (AF-1), in the N-terminal domain that is essential for AR transactivation, we evaluate the ability of EPI to overcome several clinically relevant AR-related mechanisms of resistance. Experimental Design: To study the effect of EPI on AR transcriptional activity against overexpressed coactivators, such as SRC1-3 and p300, luciferase reporter assays were performed using LNCaP cells. AR-negative COS-1 cells were employed for reporter assays to examine whether the length of polyglutamine tract affects inhibition by EPI. The effect of EPI on constitutively active AR splice variants was studied in LNCaP95 cells, which express AR-V7 variant. To evaluate the effect of EPI on the proliferation of LNCaP95 cells, we performed in vitro BrdUrd incorporation assay and in vivo studies using xenografts in mice. Results: EPI effectively overcame several molecular alterations underlying aberrant AR activity, including overexpressed coactivators, AR gain-of-function mutations, and constitutively active AR-V7. EPI inhibited AR transcriptional activity regardless of the length of polyglutamine tract. Importantly, EPI significantly inhibited the in vitro and in vivo proliferation of LNCaP95 prostate cancer cells, which are androgen independent and enzalutamide resistant. Conclusions: These findings support EPI as a promising therapeutic agent to treat CRPC, particularly against tumors driven by constitutively active AR splice variants that are resistant to LBD-targeting drugs. Clin Cancer Res; 22(17); 4466–77. ©2016 AACR. See related commentary by Sharp et al., p. 4280

Characterization of Niphatenones that Inhibit Androgen Receptor N-Terminal Domain

Androgen ablation therapy causes a temporary reduction in tumor burden in patients with advanced prostate cancer. Unfortunately the malignancy will return to form lethal castration-recurrent prostate cancer (CRPC). The androgen receptor (AR) remains transcriptionally active in CRPC in spite of castrate levels of androgens in the blood. AR transcriptional activity resides in its N-terminal domain (NTD). Possible mechanisms of continued AR transcriptional activity may include, at least in part, expression of constitutively active splice variants of AR that lack the C-terminal ligand-binding domain (LBD). Current therapies that target the AR LBD, would not be effective against these AR variants. Currently no drugs are clinically available that target the AR NTD which should be effective against these AR variants as well as full-length AR. Niphatenones were originally isolated and identified in active extracts from Niphates digitalis marine sponge. Here we begin to characterize the mechanism of niphatenones in blocking AR transcriptional activity. Both enantiomers had similar IC50 values of 6 µM for inhibiting the full-length AR in a functional transcriptional assay. However, (S)-niphatenone had significantly better activity against the AR NTD compared to (R)-niphatenone. Consistent with niphatenones binding to and inhibiting transactivation of AR NTD, niphatenones inhibited AR splice variant. Niphatenone did not affect the transcriptional activity of the related progesterone receptor, but slightly decreased glucocorticoid receptor (GR) activity and covalently bound to GR activation function-1 (AF-1) region. Niphatenone blocked N/C interactions of AR without altering either AR protein levels or its intracellular localization in response to androgen. Alkylation with glutathione suggests that niphatenones are not a feasible scaffold for further drug development.

Sintokamide A Is a Novel Antagonist of Androgen Receptor That Uniquely Binds Activation Function-1 in Its Amino-terminal Domain

Androgen receptor (AR) is a validated drug target for all stages of prostate cancer including metastatic castration-resistant prostate cancer (CRPC). All current hormone therapies for CRPC target the C-terminal ligand-binding domain of AR and ultimately all fail with resumed AR transcriptional activity. Within the AR N-terminal domain (NTD) is activation function-1 (AF-1) that is essential for AR transcriptional activity. Inhibitors of AR AF-1 would potentially block most AR mechanisms of resistance including constitutively active AR splice variants that lack the ligand-binding domain. Here we provide evidence that sintokamide A (SINT1) binds AR AF-1 region to specifically inhibit transactivation of AR NTD. Consistent with SINT1 targeting AR AF-1, it attenuated transcriptional activities of both full-length AR and constitutively active AR splice variants, which correlated with inhibition of growth of enzalutamide-resistant prostate cancer cells expressing AR splice variants. In vivo, SINT1 caused regression of CRPC xenografts and reduced expression of prostate-specific antigen, a gene transcriptionally regulated by AR. Inhibition of AR activity by SINT1 was additive to EPI-002, a known AR AF-1 inhibitor that is in clinical trials (NCT02606123). This implies that SINT1 binds to a site on AF-1 that is unique from EPI. Consistent with this suggestion, these two compounds showed differences in blocking AR interaction with STAT3. This work provides evidence that the intrinsically disordered NTD of AR is druggable and that SINT1 analogs may provide a novel scaffold for drug development for the treatment of prostate cancer or other diseases of the AR axis.

Abstract B14: Developing small-molecule inhibitors to the androgen receptor N-terminus domain for the treatment of advanced prostate cancer

Abstract Androgen ablation therapy remains the gold standard for the treatment of advanced prostate cancer, but unfortunately, it is not curative and eventually the disease will return as lethal castration-resistant prostate cancer (CRPC). There is evidence supporting the concept that development of CRPC is causally related to continued transactivation of androgen receptor (AR). Suspected mechanisms for continued AR activity in spite of castrate levels of androgen include: amplification or overexpression of AR; gain-of-function mutations allowing AR to be activated by steroids or antiandrogens; ligand-independent activation by growth factors, cytokines, or kinases; overexpression of AR coactivators; intracrine signaling by increased intratumoral androgens; and/or expression of constitutively active splice variants of AR that lack the C-terminal ligandbinding domain (LBD). All current therapies that target the AR are dependent on the presence of its C-terminal LBD. However, it is the N-terminal domain (NTD) of the AR that is the “Achilles Heel” of AR activity, with activation function-1 (AF-1) being essential for AR activity regardless of androgen. Our efforts have been focused upon developing drugs to the AR NTD and have yielded EPI-001 a small molecule, sintokamide peptides, and decoys to the AR NTD. Of these, EPI-001 is the best characterized as previously shown to inhibit essential protein-protein interactions that are required for AR transcriptional activity. EPI-001 and its analogues (generally referred to as “EPI”) have great promise for clinical development based upon its unique mechanism of action, specificity, low toxicity, and cytoreductive antitumor activity. EPI blocked transcriptional activity of full-length and AR variants as well as specifically inhibited AR-dependent cell proliferation. EPI directly and specifically interacted with AF1 and did not interact with denatured AF1 as shown using in vitro binding assays. Specific and direct interaction of EPI with the endogenous AR occurred in living cells as shown using click chemistry. EPI-001 had 86% oral bioavailability, a half-life of 3.4 hours, and plasma levels at the effective concentration of 10 ug/ml were achieved with oral dosing. Consistent with excellent oral bioavailability, oral dosing of EPI inhibited VCaP tumor growth in castrated animals. VCaP human prostate cancer cells express an abundance of full-length AR as well as constitutively active AR splice variant lacking LBD. Evidence for EPI targeting the AR transcriptional program in vivo, was provided by reduced transcripts of UBE2C, CDC20, cyclinA2, and AKT1 in harvested VCaP xenografts from animals treated orally with EPI. In conclusion, EPI is an antagonist of AR NTD that blocks the activity of AR, including constitutively active AR splice variants, by a mechanism that involves direct interaction with the NTD. Oral dosing of EPI has antitumor activity in prostate cancer xenografts that express AR variant. Together these data support the clinical development of EPI for the treatment of CRPC. Funding: NIH (2R01 CA105304) and US Army Medical Research and Materiel Command Prostate Cancer Research Program (PC100761). Note: This abstract was not presented at the conference because the presenter was unable to attend. Citation Format: Marianne D. Sadar, Jae-Kyung Myung, Iain McEwan, Stephen Plymate, Raymond J. Andersen, Carmen A. Banuelos, Nasrin R. Mawji, Jun Wang, Javier Garcia Fernandez, Amy Tien, Iran Tavakoli, Yu Chi Yang, Simon Haile. Developing small-molecule inhibitors to the androgen receptor N-terminus domain for the treatment of advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B14.