开发 HiBiT 标记的高通量筛选实验以发现前列腺癌中靶向 SKP2 的降解剂

S-phase kinase associated protein 2 (SKP2), an E3 ubiquitin ligase for protein degradation and regulating the cell cycle, is overexpressed in high-grade prostatic intraepithelial neoplasia and prostate adenocarcinoma compared to normal prostate tissues, and its higher mRNA levels in prostate tumor tissues are significantly associated with poorer survival of prostate cancer patients. Therefore, identifying novel molecules that promote SKP2 degradation represents a promising strategy for intercepting prostate cancer. We have generated the CRISPR/Cas9 knock-in (KI) of the HiBiT tag at the endogenous SKP2 locus in the 22Rv1 prostate cancer cell line with SKP2 gene amplification to enable high-throughput screening of SKP2 protein degraders. Sanger sequencing, western blot and luminescence imaging were performed to confirm the successful expression of the endogenous SKP2-HiBiT in 22Rv1 cells. Screening conditions adapted with nano-Glo® HiBiT lytic detection assay were well established in a 384 well plate format, yielding a high signal-to-background ratio of 69. We have further validated the screening assay with SKP2 C1 inhibitor and Flavokawain A, a naturally occurring chalcone from the Kava plants. Flavokawain A (degrading SKP2 protein) but not C1 (a SKP2 enzyme inhibitor) inhibited the HiBiT luminescence signal, confirming the specificity of the assay. In addition, we have synthesized a small library of chalcone derivatives and by screening them in SKP2-HiBiT-KI 22Rv1 cells, three new SKP2 degraders with improved efficacy have been identified. To avoid false positives due to assay artifacts, compounds that significantly inhibit luminescence within 5 minutes of treatment are excluded from the hits list. The screening assay exhibited robust performance with Z′ factors exceeding 0.6. These three SKP2 degraders significantly reduced the HiBiT luminescent signal at low micromolar concentrations after 8 hours of treatment compared to vehicle control treatment, with minimal effects on cell viability. Western blot analyses further confirmed that these three hits downregulated the protein levels of SKP2 in a dose and time dependent manner. In addition, PC3 cells overexpressing SKP2 are more sensitive to the growth inhibitory effects of these three degraders than SKP2 non-overexpressing PC3 control cells, suggesting the specificity of these degraders for targeting SKP2 overexpressing prostate cancer cells. In conclusion, we successfully established and validated the SKP2-HiBiT-KI 22Rv1screening platform that enables the identification of highly potent, low-toxicity SKP2 degraders for prostate cancer interception.