Ligand-specific binding kinetics and molecular interactions of clinically used PSMA diagnostic radiopharmaceuticals: an integrated in silico and in vitro study

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Abstract Background Prostate-specific membrane antigen (PSMA)–targeting radiopharmaceuticals are widely used for positron emission tomography (PET) imaging of prostate cancer and are often grouped as a single diagnostic class in clinical guidelines. However, beyond PSMA selectivity, each radiopharmaceutical exhibits distinct in vivo behaviour, and the molecular interactions and binding kinetics underlying these differences remain incompletely understood. This study aimed to define the mechanistic determinants of ligand-specific behaviour among clinically used 18 F-fluorinated radiotracers by integrating molecular simulations with real-time cellular binding assays. Molecular docking and molecular dynamics simulations were used to characterise the binding of the fluorinated PSMA radiopharmaceuticals [ 18 F]PSMA-1007, [ 18 F]DCFPyL, and [ 18 F]JK-PSMA-7 within the PSMA active site, with particular attention to zinc coordination and water-mediated interactions. Relative binding energies were estimated using the Molecular Mechanics Poisson–Boltzmann Surface Area method. Real-time cellular binding kinetics were assessed using the LigandTracer system to estimate apparent association and dissociation rate constants. These experiments included the three fluorinated tracers and [ 68 Ga]Ga-PSMA-11 as a widely used clinical reference. Competition assays with 2-(phosphonomethyl)pentanedioic acid were also performed. Results All three fluorinated radiopharmaceuticals investigated in silico bound within the PSMA active site through the conserved Glu–urea–Lys motif and maintained stable interactions with the binuclear zinc centre. Molecular simulations revealed ligand-specific differences in the occupation of the active-site funnel and showed substantial contributions from water-mediated hydrogen-bond networks, particularly for [ 18 F]PSMA-1007. In cellular experiments, [ 68 Ga]Ga-PSMA-11 exhibited the highest mean apparent association rate and the most pronounced decrease in cell-associated signal during the dissociation phase. In contrast, [ 18 F]PSMA-1007 showed the lowest mean apparent association rate and no measurable signal decrease during the experimental dissociation period, whereas [ 18 F]DCFPyL and [ 18 F]JK-PSMA-7 displayed intermediate kinetic profiles. Competition assays yielded progressively higher apparent IC 50 estimates from [ 68 Ga]Ga-PSMA-11 to [ 18 F]PSMA-1007. No measurable decrease in [ 18 F]PSMA-1007-associated signal was observed during the 30-minute real-time competition experiment, even at the highest inhibitor concentration. Conclusion Despite sharing a conserved PSMA-binding motif, clinically used PSMA radiopharmaceuticals exhibited distinct molecular interaction patterns and apparent cellular binding kinetics under the experimental conditions examined, taking into account the simplifications adopted for the design of the study and for the analysis of the results. These findings provide a mechanistic basis for tracer-specific behaviour and support further investigation of whether such differences influence clinical imaging characteristics.

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