Open Collections

Abstract

Background: Current therapies for prostate cancer (PCa) involve pharmaceutical castration by targeting the androgen receptor (AR) ligand-binding domain (LBD). These therapies are not curative with the malignancy progressing to castration-resistant PCa (CRPC). Most CRPC remains driven by the AR through multiple resistance mechanisms, e.g., constitutively active AR splice variants that lack LBD (AR-V7), gain-of-function mutations in LBD, or AR overexpression. This has led to the clinical development of EPI analogs (“EPI”) which target the AR N-terminal domain (NTD) to block the transcriptional activities of AR-V7 and mutated ARs. The EPI-binding pocket of the AR-NTD is enriched in cysteine residues. We hypothesize that cysteine residues may alter AR transcriptional activity and impact the binding mechanism of EPI analogs. Here we begin to elucidate the role of cysteines in the inhibitory mechanism of EPI on AR activity. Methods: Site-directed mutagenesis was employed to generate full-length AR (FL-AR) and AR-V7 expression vectors containing point mutations at C264 and C509. FL-AR plasmids were co-transfected into CV-1 or PC-3 cells with AR-driven luciferase reporters to assess transcriptional activity. AR-V7 plasmids were co-transfected into LNCaP cells with an AR-V7 specific reporter. IC50 values were determined for a range of AR inhibitors to assess cysteine mutation’s impact. Results: Cysteine mutations in FL-AR led to reporter-specific and cell-specific increases in transcriptional activity in response to androgens. Significant increases were seen in CV-1 cells with ARR3tk-luc activity in both C264A and C509A mutations. There were no significant IC50 differences between AR-WT and cysteine-mutant constructs for EPI analogs, except for AR-V7 with EPI-7386, in which both mutations raised the IC50, suggesting decreased potency. As expected, the AR-LBD inhibitor enzalutamide was not able to inhibit AR-V7 activity. Conclusion: Cysteine mutations at the AR-NTD potentially play a role in modulating AR transcriptional activity and the binding capabilities of EPI analogs. Future studies are needed to explore additional cysteine residues within the AR-NTD to further elucidate their impact on transcriptional regulation and EPI binding potential. Ultimately, these findings provide valuable insights into the development of novel therapeutic strategies for advanced prostate cancer, particularly in cases where current treatments are ineffective.