Developing a Fluorescence-Based High-Throughput Screening Method for Natural Product Fractions to Identify Inhibitors...

Background/Objectives: The progression of prostate cancer to castration-resistant prostate cancer (CRPC) is often driven by constitutively active androgen receptor splice variants, such as AR-V7, which evade conventional androgen-deprivation therapies. This study aimed to develop a high-throughput, mechanism-based screening platform to identify natural-product-derived AR-V7 inhibitors from the NCI Program for Natural Product Discovery (NPNPD) library. Methods: A subset of prefractionated NPNPD samples was screened using a CRISPR-edited 22Rv1 cell line expressing endogenous AR-V7 fused to a HiBiT luminescent tag, enabling quantification of AR-V7 levels. Fractions that reduced the HiBiT signal were further evaluated in 22Rv1 and C4-2B CRPC cells and counter-screened in non-malignant RWPE-1 cells. Compounds demonstrating at least 90% inhibition in CRPC cells with no more than 10% toxicity in RWPE-1 cells underwent dose-response analysis, Western blotting, quantitative PCR, and subfractionation to isolate and characterize active constituents. Results: Of the 704 prefractionated samples, five fractions met stringent activity and selectivity criteria, with three consistently suppressing full-length androgen receptor (AR), AR-V7, and prostate-specific antigen (PSA). Primary screening results in HiBiT-22Rv1 cells were validated by secondary dose-response and Western blot assays, and hits were further prioritized to ensure toxicity remained at or below 10% in RWPE-1 cells. HiBiT-guided screening of 66 subfractions identified seven that robustly downregulated AR signaling, with reduced AR-V7 levels correlating with decreased CRPC cell viability. Chemical characterization revealed two active butanolides, isolitsealiicolide C and isolinderanolide B, which reduced AR and AR-V7 protein and mRNA levels, decreased PSA, downregulated Bcl-2, and induced cleaved PARP, consistent with apoptotic cell death in AR-positive CRPC models. Conclusions: This integrated high-throughput workflow efficiently identifies AR-V7-targeted natural products from complex libraries and highlights isolitsealiicolide C and isolinderanolide B as promising scaffolds for overcoming androgen receptor-driven resistance in CRPC.