Evaluation of the reducing potential of PSMA-containing endosomes by FRET imaging

Abstract

Aim: Ligand-targeted therapeutics are experiencing increasing use for treatment of human diseases due to their ability to concentrate a desired drug at a pathologic site while reducing accumulation in healthy tissues. For many ligand-targeted drug conjugates, a critical aspect of conjugate design lies in engineering release of the therapeutic payload to occur only after its internalization by targeted cells. Because disulfide bond reduction is frequently exploited to ensure intracellular drug release, an understanding of the redox properties of endocytic compartments can be critical to ligand-targeted drug design. While the redox properties of folate receptor trafficking endosomes have been previously reported, little is known about the trafficking of prostate-specific membrane antigen (PSMA), a receptor that is experiencing increasing use for drug targeting in humans. Methods: To obtain this information, we have constructed a PSMA-targeted fluorescence resonance energy transfer pair that reports on disulfide bond reduction by changing fluorescence from red to green. Results: We show here that this reporter exhibits rapid and selective uptake by PSMA-positive cells, and that reduction of its disulfide bond proceeds steadily but incompletely following internalization. The fact that maximal disulfide reduction reaches only ~50%, even after 24 h incubation, suggests that roughly half of the conjugates must traffic through endosomes that display no reducing capacity. folate trafficked conjugates, it also follows that not all internalizing receptors are translocated through similar intracellular compartments. Taken together, these data suggest that the efficiency of disulfide bond reduction must be independently analyzed for each receptor trafficking pathway when disulfide bond reduction is exploited for intracellular drug release.

Selective targeting of chemically modified miR-34a to prostate cancer using a small molecule ligand and an endosomal escape agent

“…At both 4 and 8 h after treatment, DUPA-miR-34a-Atto.647N conjugates co-localized with LysoTracker, suggesting sequestration into late endosomes or lysosomes ( Figures 2 G and 2H), as previously reported. 36 , 37 …”

FAP Radioligand Linker Optimization Improves Tumor Dose and Tumor-to-Healthy Organ Ratios in 4T1 Syngeneic Model

“…This correlation is notably not true for radioligands that target the PSMA receptor in which albumin binders of moderate strength (e.g., toluene, chlorophenyl, etc.) can facilitate tumor saturation equally as well as strong albumin binders (e.g., iodophenyl). , This difference in the PK-to-tumor dose relationship may be due to the slower internalization and recycling kinetics of FAP compared to PSMA. ,− Additionally, the binding affinity of the FAP6 radioligands did not seem to directly correlate with the initial tumor uptake, the long-term tumor retention, or healthy tissue clearance. While some have postulated that subnanomolar binding affinities can be harmful to the performance of antibody–drug conjugates because of a phenomenon termed the binding site barrier effect, which may result in inferior tumor penetration, it is unclear whether this hypothesis has any merit for small molecule targeting ligands. − In summary, the dosimetry calculations confirm that FAP6-19 possesses differentiated PK/PD properties from the other six derivatives because of the linker modifications.…”