The androgen receptor (AR) plays a predominant role in prostate cancer (PCa) pathology. It consists of an N-terminal domain (NTD), a DNA-binding domain (DBD), a hinge region (HR), and a ligand-binding domain (LBD) that binds androgens, including testosterone (T) and dihydrotestosterone (DHT). Ligand binding at the LBD promotes AR dimerization and translocation to the nucleus where the DBD binds target DNA. In PCa, AR signaling is perturbed by excessive androgen synthesis, AR amplification, mutation, or the formation of AR alternatively spliced variants (AR-V) that lack the LBD. Current therapies for advanced PCa include androgen synthesis inhibitors that suppress T and/or DHT synthesis, and AR inhibitors that prevent ligand binding at the LBD. However, AR mutations and AR-Vs render LBD-specific therapeutics ineffective. The DBD and NTD are novel targets for inhibition as both perform necessary roles in AR transcriptional activity and are less susceptible to AR alternative splicing compared to the LBD. DBD and NTD inhibition can potentially extend patient survival, improve quality of life, and overcome predominant mechanisms of resistance to current therapies. This review discusses various small molecule and other inhibitors developed against the DBD and NTD—and the current state of the available compounds in clinical development.

Background: The androgen receptor (AR), which plays a major role in prostate cancer (PCa), consists of a C-terminal ligand-binding domain (LBD), a hinge domain, a DNA-binding domain (DBD), and an N-terminal domain (NTD). Current AR antagonists and AR signaling inhibitors (ASIs), used in androgen deprivation therapy (ADT) and to prevent disease progression, often fail due to production of alternatively spliced AR variants (AR-Vs) that lack the LBD but retain the DBD, which is essential to AR transcriptional activity. This project examines a series of novel compounds designed to inhibit AR-DNA interaction and study how these compounds affect castration-resistant PCa (CRPC). Methods: Compounds potentially targeting the DBD were used to predict direction and position of compounds when docked to the AR. MTT assays, flow cytometry, immunoblots, qRT-PCR, luciferase assay, and immunofluorescence were used to test compounds on AR and PCa. Drug Affinity Responsive Target Stability (DARTS) tested potential binding of the compounds to the AR. Studies in intact mice were used to determine toxicity of the compounds and the maximum tolerated dose. Results: Of 48 compounds synthesized, C15 had the highest and most consistent efficacy overall. C15 decreased metabolic activity in 22Rv1 and CWR-R1 cells that express AR-Vs and in C4 and C4-2 that do not express AR-Vs. Further, C15 had a reduced effect in AR-null PC3 cells and minimal effect in normal human dermal fibroblast (NHDF) cells. In C4-2 and 22Rv1, C15 significantly reduced PSA mRNA and AR transcriptional activity, and in C4-2, C15 significantly reduced TMPRSS2 and Nrdp1 mRNA levels. C08 and C15 reduced pronase-induced AR proteolysis only when the AR DBD is present. Furthermore, C15 reduces AR binding at the PSA enhancer, but DHT stimulation may weaken this binding inhibition and reduction in AR transcriptional activity. However, combining C15 with an AR inhibitor targeting the LBD (enzalutamide, darolutamide, apalutamide) restores the full inhibitory potential and even reduces metabolic activity and AR transcriptional activity further than either drug alone in both C4-2 and 22Rv1s. No toxicity in intact mice was observed with C15 with the highest dose tested being 50 mg/kg. Conclusion: Our preliminary data demonstrate that C15 directly binds to the DBD of the AR and potently inhibits CRPC cells, including those that express AR-Vs missing the LBD. Although increased DHT levels may weaken C15’s inhibition of the AR DBD, addition of a LBD-targeting AR inhibitor improves the inhibitory potential of both drugs. Based on these results, we conclude that C15 may hold great clinical potential as it successfully inhibits CRPC by targeting the AR DBD with minimal toxicity, and it can improve the effects of standard-of-care LBD-targeting AR inhibitors by reducing AR-V-induced resistance. Citation Format: Thomas M. Steele, Elisabeth A. Messner, Maria Malvina Tsamouri, Salma Siddiqui, Brian Bennion, Ruiwu Liu, Paramita Ghosh. Targeting the DNA-binding domain of the androgen receptor in castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2650.