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Page 224 Venkatesh et al. Cancer Drug Resist 2021;4:223-32 I http://dx.doi.org/10.20517/cdr.2020.84

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, prostate-specific membrane antigen, endosomes, endocytosis

INTRODUCTION
As increasing emphasis is placed on precision medicine, ligand-targeted therapies are becoming
[1,2]
increasingly important in treating human diseases . Advantages of ligand-targeted therapies are manifold,
including reduced toxicities to healthy tissues, increased accumulation and retention of drug in pathologic
tissues, the capability to administer therapeutic agents that would not be tolerated in non-targeted form,
and the ability to select responsive patients using a companion diagnostic comprised of the targeting ligand
[3]
linked to an imaging agent . These targeted therapeutics typically contain a ligand that specifically binds
to an upregulated receptor on the pathologic cell surface, a linker region that can be modified to optimize
[4]
the physical and chemical properties of the conjugate, and a therapeutic payload . Unsurprisingly, the
design of these conjugates to assure that they remain intact while circulating throughout the body and
then reproducibly unload their cargoes after uptake by their target cells is often critical to both safety and
efficacy.

While the release of the therapeutic payload of some ligand-targeted drugs is not desired (e.g.,
[5]
radioisotopes), other conjugates require discharge of their payloads to be efficacious . For these ligand-
drug conjugates, a mechanism that has been frequently exploited for intracellular drug release has involved
[6,7]
cleavage of a disulfide bond . This preference has arisen from the fact that intracellular environments are
largely reducing, whereas extracellular environments such as the bloodstream and interstitial spaces are
[8]
largely oxidizing . The above difference in redox potential has been hypothesized to allow for reduction
of a disulfide bond within intracellular compartments that would normally remain intact during transit
through extracellular environments. Curiously, although the internalization pathways for many cell surface
receptors have been characterized (i.e., pinocytosis, clathrin-coated pit mediated endocytosis, potocytosis,
micropinocytosis, etc. [9,10] ), little if any information exists on whether all or only a fraction traffic to a
reducing environment.

In an earlier report, the intracellular reducing potential of a cancer cell was examined using a folate-
targeted conjugate containing a fluorescence resonance energy transfer (FRET) pair in which reduction
of a disulfide bond connecting the donor and acceptor dyes led to dequenching of the donor fluorophore
[11]
and the consequent appearance of its fluorescence . Using this strategy, the rates of disulfide bond
reduction following conjugate internalization and trafficking through various endosomal compartments
were quantifiable. It was found that disulfide bond reduction began immediately upon endocytosis,
continued during intracellular trafficking and approached completion by 12 h after administration .
[11]
While this information has proven useful in designing the release of folate-targeted therapeutic agents [12,13] ,
the question has always remained whether the kinetics of disulfide bond reduction in the folate receptor
endocytic pathway can be extrapolated to release of ligand-targeted drugs that enter cells and traffic
through other endocytic pathways.

As numerous prostate specific membrane antigen (PSMA)-targeted drugs and imaging agents have been
studied in both animals [14-16] and humans bearing prostate cancers [17-19] , we elected to explore this question