Site-specific conjugation of PEG12-PSMA ligand to dianthin by click chemistry to kill prostate cancer cells in the presence of the endosomal escape enhancer SO1861.

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Background: Prostate-specific membrane antigen (PSMA) is overexpressed in many prostate cancer cells and is a promising target for the diagnosis and treatment of prostate cancer. This study aims to develop a novel PSMA-targeted ligand-toxin conjugate that kills prostate cancer cells by ribosomal inactivation, and to evaluate its potency in vitro . Methods: A modified single-cysteine-containing ribosome-inactivating protein (Dianthin-Cys) was recombinantly expressed in NiCo21 cells and purified by Ni-NTA column chromatography followed by cation exchange chromatography. An azido-PSMA ligand was site-specifically coupled to Dianthin-Cys by click chemistry via a maleimide-PEG12-DBCO linker (D-PSMA). Ribosomal N-glycosidase activity of Dianthin-Cys was measured by an adenine-release assay. Cytotoxicity was assessed with or without the endosomal escape enhancer SO1861 to determine IC ₅₀ values in PSMA-positive C4-2 and LNCap cells and in PSMA-negative DU145 and PC-3 cells. Fluorescently labeled D-PSMA was used for confocal microscopy to visualize surface binding and internalization of the conjugates in each cell line. Lysosomal trafficking was assessed with LysoTracker Green DND-26. Results: SDS-PAGE and mass spectrometry indicated high purity of D-PSMA conjugates, with a molecular mass of ~32 kDa. Western blotting confirmed strong PSMA expression in C4-2 and LNCap cells but not in DU145 and PC-3 cells. The adenine release activity of D-PSMA was 71.93 pmol/pmol/h. Cytotoxicity assays showed prominent cell death in the presence of SO1861, with IC ₅₀ values of 0.738 pM and 0.695 pM in C4-2 and LNCap cells, respectively, and 55 pM and 26 pM in DU145 and PC-3 cells, respectively. No cytotoxicity was observed in the absence of SO1861 within the tested concentration range (up to 3.1 nM). Prominent surface binding and internalization of D-PSMA were observed in C4-2 and LNCap cells, whereas DU145 and PC-3 cells showed no visible binding or internalization. Both C4-2 and LNCap cells showed colocalization of D-PSMA with Lysotracker. Conclusions: The findings of this study demonstrate that D-PSMA is a PSMA-specific and effective therapeutic agent when combined with SO1861 for the treatment of prostate cancer.