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.
Highlights
As increasing emphasis is placed on precision medicine, ligand-targeted therapies are becoming increasingly important in treating human diseases[1,2]
As the level of disulfide reduction differs between prostate-specific membrane antigen (PSMA) trafficked and previously published folate trafficked conjugates, it follows that not all internalizing receptors are translocated through similar intracellular compartments
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
Summary
As increasing emphasis is placed on precision medicine, ligand-targeted therapies are becoming increasingly important in treating human diseases[1,2]. 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 linked to an imaging agent[3] These targeted therapeutics typically contain a ligand that binds to an upregulated receptor on the pathologic cell surface, a linker region that can be modified to optimize the physical and chemical properties of the conjugate, and a therapeutic payload[4]. While the release of the therapeutic payload of some ligand-targeted drugs is not desired (e.g., radioisotopes), other conjugates require discharge of their payloads to be efficacious[5] For these liganddrug conjugates, a mechanism that has been frequently exploited for intracellular drug release has involved cleavage of a disulfide bond[6,7]. 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
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