Javier Garcia Fernandez

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).

Abstract 2661: Novel albumin-binding maytansinoids inducing long-term partial and complete tumor regressions in several human cancer xenograft models in nude mice

MDA-MB 468 Maytansine and its analogs (e.g. DM1 and DM4) are potent microtubule-targeting compounds with a narrow therapeutic window. So far, only T-DM1, an antibody-maytansinoid conjugate targeting the HER2 receptor, has been approved for the treatment of Herceptin®-resistant breast cancer. Our design of two novel albumin-binding maytansinoids (LADR-9 and LADR-10) is based on: • Identification of two novel maytansine-based highly potent payloads (ANSA-05, ANSA-13), selected from screening a library of maytansinoids in vitro (Poster #1657) • Derivatization with a new water-solubilizing linker (SULF-07) resulting in LADR-9 and LADR-10 which bind in situ to the Cys-34 position of endogenous albumin • Accumulation of the drug-albumin conjugate in tumor tissue • Acid-mediated drug release at the tumor site INTRODUCTION & RATIONALE

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.