Computational and Experimental Studies of PSMA-Targeted Conjugates Designed for Subcellular Delivery of Theranostic Radiometals to Prostate Cancer Cells

Abstract Background: The membrane receptor prostate-specific membrane antigen (PSMA) is a validated target for theranostic radioconjugates in the treatment and imaging of prostate cancer (PCa). Radioconjugates targeted at PSMA rely mostly on the peptidomimetic glutamate–urea–lysine (Glu–urea–Lys) binding motif to facilitate tumor accumulation. The combination of this binding motif with subcellular targeting moieties, such as acridine orange (AO) and triphenylphosphonium (TPP) derivatives, for nuclear and mitochondrial delivery, respectively, might open the way to innovative dual-target approaches. This study integrated molecular docking, long-timescale molecular dynamics (MD) simulations, and in vitro PSMA inhibition assays to assess whether the incorporation of AO or TPP disrupts the canonical PSMA binding in DOTA-based conjugates containing the Glu–urea–Lys binding motif designed by our group. Results: Docking analyses indicated that our PSMA-targeted conjugates carrying AO or TPP moieties adopt poses analogous to those of PSMA’s natural substrate N-acetyl-aspartyl-glutamate (NAAG) and of the reference conjugate natIn-PSMA-617, preserving crucial interactions within the arginine-rich S1 pocket and within the glutamate-sensing S1′ pocket. In contrast, their congeners lacking the Glu–urea–Lys binding motif exhibited surface-associated binding but failed to engage stably within the active site of PSMA. MD simulations confirmed the structural stability of the PSMA-conjugate complexes. In vitro inhibition assays supported these observations: the non-PSMA-targeted natIn-AO and natIn-TPP displayed negligible inhibitory activity against PSMA (with noncalculable IC50 values), whereas the dual-target conjugates displayed potency in the submicromolar range. Conclusion: Collectively, our results show that the dual-target conjugates recognize the PSMA protein, with subcellular targeting motifs influencing peripheral dynamics, but not core binding. This framework supports the rational design of multifunctional PSMA-directed radioconjugates for improved subcellular delivery of theranostic radionuclides toward alternative In PCa imaging and management.