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Venkatesh et al. Cancer Drug Resist 2021;4:223-32 I http://dx.doi.org/10.20517/cdr.2020.84 Page 229

the conjugate was quantitatively cleaved in the presence of a reducing agent, thus confirming the
susceptibility of the disulfide bond to reducing conditions.

PSMA-mediated internalization of the DUPA-FRET conjugate
Next, to ensure that the DUPA-FRET conjugate would be selectively internalized by PSMA-mediated
endocytosis, the conjugate was incubated for 1 hour with either a PSMA-negative (PC-3 cells) or PSMA-
[22]
positive (LNCaP cells) prostate cancer cell line . As shown in Figure 1D, internalization of the conjugate
only occurred in the PSMA-positive LNCaP cells, demonstrating that PSMA is essential for DUPA-FRET
uptake by prostate cancer cells. Additionally, when the cells were excited with a green (488 nm) laser,
mostly red fluorescence was observed, confirming that the conjugate facilitated efficient FRET within the
cells.

Fluorescence changes of the DUPA-FRET conjugate following endocytosis
With the fluorescent properties of the PSMA-targeted DUPA-FRET conjugate confirmed, the reducing
potential of PSMA-trafficking endosomes was evaluated by incubating LNCaP cells in the presence of the
conjugate for various times and measuring the fluorescent signals from both sulforhodamine B (i.e., red
FRET signal of an intact conjugate) and Bodipy (i.e., green signal of a reduced conjugate). As shown in
Figure 2, the red FRET signal predominated during the first two hours when the cells were excited at 488 nm,
with the vast majority of fluorescence localized to intracellular endosomes even by the 1 h time point (panel
2B). However, as time proceeded, the red FRET signal declined while the green signal increased, indicating
that disulfide bond reduction was occurring and the donor/acceptor FRET pair were separating. That the
decrease in red fluorescence during 488 nm excitation did not derive from destruction of the rhodamine
fluorophore was established by demonstrating that direct excitation of the rhodamine dye with 568 nm
light revealed no diminution of fluorescence (data not shown). Interestingly, however, changes in the FRET
signal indicated that cleavage of the disulfide bond proceeded to only ~50% completion, even after 24 h
incubation [Figure 3], suggesting that approximately half of the DUPA-FRET conjugate must traffic to an
endosomal compartment in which the reducing environment is insufficient to cleave a disulfide bond.

DISCUSSION
In conclusion, information on the internalization and reduction of disulfide bonds can be critical to
the design of ligand-targeted drugs, especially in cases where targeting ligand and drug are connected
by a disulfide bond [23,24] . However, while PSMA is a commonly used receptor for such ligand-targeted
[26]
therapeutic agents [14-19,25] , and recycling of the PSMA receptor has been studied , the kinetics of disulfide
bond cleavage in PSMA-containing intracellular compartments has not been thoroughly explored.
Therefore, in order to measure these kinetics and gain a greater understanding of the reducing environment
within PSMA-trafficking endosomes, a PSMA-targeted FRET-based probe containing the DUPA-targeting
ligand linked to a disulfide-bridged FRET donor/acceptor pair was synthesized.

This PSMA-targeted drug conjugate was intentionally designed to contain membrane impermeable
fluorescent reporter molecules. Thus, the green fluorescent dye is attached to the DUPA ligand via a
non-cleavable linker in order to allow the investigator to track where the DUPA ligand traffics following
its endocytosis by the cancer cell. Similarly, the red fluorescent dye was also designed to be membrane
impermeable to allow the researcher to identify and monitor the endosomes in which the released drug
would normally traffic. Thus, we intentionally made our fluorescent reporter molecules membrane
impermeable in order to enable us to track their itineraries within the cancer cell. However, if we were to
prepare a prostate cancer targeted therapeutic drug, it would be designed to be membrane permeable so
that when it was released within an intracellular endosome, it would be able to diffuse out of the endosome
and engage its therapeutic target elsewhere in the cell.