针对雄激素受体的第二代AUTOTAC用于去势抵抗性前列腺癌治疗的表征研究

Background: The androgen receptor (AR) drives the progression of prostate cancer (PCa), including castrate-resistant PCa (CRPC). Therapeutic resistance is often mediated by AR mutations and splice variants like AR-v7, which circumvent conventional AR-targeting therapies. AUTOTACs (Autophagy-Targeting Chimeras) is a novel platform for targeted protein degradation via the autophagy-lysosomal pathway. We previously reported the characterization of ATC-324, the first-generation AR-targeting AUTOTAC utilizing enzalutamide (ENZ) as a target-binding ligand (TBL) and YT 6-2 as an autophagy targeting ligand (ATL). The current study evaluates two second-generation AR AUTOTACs, ATB-238 and ATB-239, designed to improve the efficacy and stability in degrading AR and its variants. Methods: AR-null PCa model: PC3. Androgen-sensitive PCa models: LNCaP, LAPC4. CRPC models: CWR-R1, 22Rv1. Immunoblotting measured relative protein levels. p62 siRNA and bafilomycin A1 were used to study autophagy-dependent drug mechanism. An AR activity reporter assay quantified AR transcriptional activity. Cytotoxicity was evaluated using the WST1 assay. Molecular docking, mutagenesis assays, and proximity ligation assay (PLA) were used to assess ATB-238- and ATB-239-induced AR:p62 complex formation. Bone-in-culture array (BICA) was used to assess the therapeutic potential of ATB-238- and ATB-239 in 22Rv1 in the bone microenvironment. Docetaxel, a first-line chemotherapy for PCa, was used to study synergy with AR AUTOTACs. Results: ATB-238 and ATB-239 demonstrated improved degradation of AR isoforms and significantly reduced AR transcriptional activity, including AR-v7-driven activity. ATB-238 demonstrated cytotoxicity against AR-positive cells (LAPC4, CWR-R1, 22Rv1), but not AR-null cells (PC3). Molecular docking predicts that ATB-238 and ATB-239 bind to R139, K141, L166, and F168 within the zz domain of p62. Mutation of these residues into Alanine abolished AR:p62 interaction and p62 oligomerization induced by ATB-239. ATB-238 and ATB-239 exhibited high potency against 22Rv1 micrometastases in BICA, while ATB-238 synergizes with docetaxel in killing LNCaP and CWR-R1 cells. Conclusion: ATB-238 and ATB-239 represent significant advances in AR-targeted therapeutics, capable of degrading AR variants and overcoming therapy resistance. The ongoing intra-iliac-artery xenograft studies will assess their potential to inhibit PCa bone metastasis, an incurable stage of the disease. These findings highlight the therapeutic promise of AUTOTAC technology in addressing critical challenges in CRPC treatment.