SAT023 The Role Of Cysteine Residues In The Binding Of Inhibitors Of The N-Terminal Domain Of The Androgen Receptor

Disclosure: L. Deneault: None. A. Tien: None. A. Banuelos: Stock Owner; Self; ESSA. T. Tam: None. R. Andersen: Stock Owner; Self; ESSA. M.D. Sadar: Stock Owner; Self; ESSA. Background: Therapies for prostate cancer (PC) 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 PC (CRPC). Most CRPC remains driven by AR through multiple resistances, e.g. constitutively active AR splice variants that lack LBD (AR-Vs), or gain-of-function mutations in LBD. This has led to clinical development of EPI analogues (“EPI”) which target the AR N-terminal domain (NTD) to block the transcriptional activities of, AR-Vs and mutated ARs. The EPI-binding pocket involves region 341-446 amino acid residues of the NTD. AR NTD is enriched in cysteine residues with C404 suggested to be important in the binding mechanism of EPI. Free cysteine residues are able interact forming disulfide bonds, which are important in intra- and intermolecular protein-protein interactions. Here we begin to elucidate the role of cysteines in the inhibition of AR activity by EPI. Methods: Binding assays assess the role of cysteines in the covalent binding mechanism used a fluorescein-labelled EPI with fragments of recombinant AR-NTD (rNTD) with or without the addition of iodoacetamide (caps cysteines). Protein-protein interactions requiring disulfide bonds with AR examined by non-reducing SDS-PAGE and diagonal SDS-PAGE of cells treated with inhibitors. A role of specific cysteines in the activity of EPI to block AR transcriptional activity was assessed by cysteine mutants using a reporter gene construct driven by AR. Results: Capping cysteine residues on rNTD with iodoacetamide reduced the amount of covalent binding of EPI. This data suggests that cysteines are essential for covalent binding of EPI to AR-NTD. EPI bound preferentially to the oxidized form of rNTD suggesting the importance of disulfide bridges. Diagonal SDS-PAGE revealed differential alterations in intramolecular and intermolecular disulfide bonds in response to EPI compared to AR LBD inhibitors. These gels also revealed unique differences in protein-protein interactions with AR depending on the inhibitor. AR-driven reporter gene assays revealed mutations of cysteine residues in the AR- NTD did not adversely affect AR transcriptional activity in response to androgen. Conclusions: Although cysteines residing in the AR-NTD were required for covalent binding of EPI, mutation of C404 in the EPI-binding site was not essential for the efficacy of EPI to block AR transcriptional activity. There were differential patterns in protein-protein interactions with AR depending upon which domain the inhibitor targeted. Our future goal is to identify these unique proteins to provide insight into the mechanisms and develop more potent and more specific therapeutics for the treatment of CRPC. Presentation: Saturday, June 17, 2023