Drug discovery targeting FOXA1 R219S mutant in advanced prostate cancer

Advanced prostate cancer is prone to recurrence and metastasis, and there are currently no effective clinical treatments available, highlighting an urgent need for the development of highly effective targeted drugs. Mutations in the transcription factor FOXA1 are significantly enriched in prostate cancers resistant to second-generation anti-androgen therapies and are highly associated with malignant tumor progression. However, no small-molecule inhibitors targeting mutant FOXA1 protein have been reported to date. This study aims to screen for targeted drugs against the FOXA1 R219S mutant protein using virtual screening combined with surface plasmon resonance (SPR) and functional validation assays such as IC50, EMSA, and RT-qPCR. By analyzing the mutation sites and frequency of FOXA1 in clinical samples, we identified hotspot mutations R219S, R219C, R261C, R261G, and R261S. Through virtual drug screening, a series of small-molecule inhibitors targeting these hotspot mutations were discovered. Among these, the R219S mutation is significantly enriched in advanced neuroendocrine prostate cancer (NEPC) and promotes malignant proliferation and metastasis of prostate cancer. Therefore, we focused on this mutation and first elucidated the binding pockets and key amino acid residues near the binding sites of drugs targeting both wild-type and R219S mutant FOXA1 proteins. To validate the binding ability of small-molecule drugs to the protein in vitro, we heterologously expressed and purified GST-FOXA1-His protein. SPR experiments confirmed that four drugs (imidazolidinyl urea, amifostine, trifluridine and asiaticoside) selectively bind to the FOXA1 R219S protein but not to the FOXA1 WT protein. Among these, the result of EMSA revealed that imidazolidinyl urea, amifostine, and trifluridine competitively inhibit the binding of FOXA1 R219S protein to DNA. CCK8 assay demonstrated that trifluridine exhibits potent cytotoxicity against prostate cancer cells (IC50=15.4 μmol/L). In summary, we have preliminarily screened drugs that selectively target FOXA1 R219S, laying the foundation for the development of targeted therapies for advanced prostate cancer.