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

Chelvam Venkatesh; Jiayin Shen; Karson S Putt; Philip S Low

doi:10.20517/cdr.2020.84

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

Cancer Drug Resist. 2021 Mar 19;4(1):223-232. doi: 10.20517/cdr.2020.84. eCollection 2021.

Authors

Chelvam Venkatesh^{1

2}, Jiayin Shen³, Karson S Putt⁴, Philip S Low^{3

4}

Affiliations

¹ Discipline of Chemistry, Indian Institute of Technology, Madhya Pradesh, Indore 453552, India.
² Discipline of Bioscience and Biomedical Engineering, Indian Institute of Technology, Madhya Pradesh, Indore 453552, India.
³ Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.
⁴ Institute for Drug Discovery, Purdue University, West Lafayette, IN 47907, USA.

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.