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. Conclusion: As the level of disulfide reduction differs between PSMA trafficked and previously published 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.

Keywords: DUPA; endocytosis; endosomes; prostate-specific membrane antigen.

Figure 1

Analysis of disulfide reduction and PSMA binding specificity of the DUPA-FRET conjugate in Scheme 1. (A) Depiction of changes of emitted fluorescence upon DUPA-FRET conjugate reduction. See Scheme 1 for color coding; (B, C) reduction of the DUPA-FRET conjugate was analyzed using RP-C18 HPLC (Abs = 280 nm) in the absence (B) and presence (C) of a 10-fold excess of dithiothreitol; (D) DUPA-FRET conjugate (100 nmol/L) was incubated with PC-3 (human PSMA-negative) and LNCaP (human PSMA-positive) cells for 1 h at 37 °C. Cells were then excited by a green (488 nm) laser and emitted green and red fluorescence were visualized via confocal microscopy

Scheme 1

Synthesis of DUPA-Lys(Bodipy FL)-S,S-Sulforhodamine FRET conjugate. Reagents and conditions: (A) Fmoc-Lys(Boc)-OH, PyBOP, DIPEA, DMF, 6 h; (B) (1) 20% piperidine in DMF, rt, 30 min; (2) Fmoc-Asp(O^tBu)-OH, PyBOP, DIPEA, DMF, 6 h; (C) (1) 20% piperidine in DMF, rt, 30 min; (2) Fmoc-Asp(O^tBu)-OH, PyBOP, DIPEA, DMF, 6 h; (D) (1) 20% piperidine in DMF, rt, 30 min; (2) Fmoc-8-aminocaprylic acid, PyBOP, DIPEA, DMF, 6 h; (E) (1) 20% piperidine in DMF, rt, 30 min; (2) DUPA(O^tBu)₃-OH, PyBOP, DIPEA, DMF, 6 h; (3) TFA/TIS/EDT/H₂O (9.25:0.25:0.25:0.25) (1 × 5 mL, 30 min; 2 × 5 mL, 5 min); (4) Evaporate TFA; (5) Precipitate in ice cold diethylether, RP-HPLC purification; (F) (1) DIPEA, DMSO, Argon, overnight; (2) RP-HPLC, 20 mM NH₄OAc, pH = 7.0, 0-50% CH₃CN in a 30-min run; (G) (1) DIPEA, DMSO, Argon, overnight; (2) RP-HPLC using 20 mmol/L NH₄OAc, pH = 7.0, 0-50% CH₃CN in a 30-min run. The final DUPA-FRET conjugate is presented in the bottom structure, where the PSMA targeting ligand (DUPA) is color-coded magenta, the peptidic spacer is color-coded blue, the fluorescence donor (Bodipy) is represented green, and the fluorescence acceptor (sulforhodamine) is colored red

Figure 2

Analysis of the kinetics of disulfide reduction in the DUPA-FRET conjugate following internationalization by LNCaP cells. (A) Cells were either left unlabeled or (B-F) labeled with DUPA-FRET (100 nmol/L) for the indicated times at 37 °C before evaluation by confocal microscopy using 488 nm excitation. A decrease in red fluorescence accompanied by an increase in green fluorescence demonstrates release of the rhodamine from the DUPA-FRET conjugate; (B) image of cells at the 1-h time point showing a punctate distribution of fluorescence throughout the cell interior; i.e., suggesting accumulation of the conjugate within intracellular compartments

Figure 3

Quantitation of DUPA-FRET fluorescence change upon internationalization in LNCaP cells. Red and green channel fluorescence intensity was quantitated and the percentage of the red compared to total fluorescence was determined and plotted. Error bars represent standard deviation. FRET: fluorescence resonance energy transfer