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Venkatesh et al. Cancer Drug Resist 2021;4:223-32 Cancer
DOI: 10.20517/cdr.2020.84 Drug Resistance

Original Article Open Access

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

Chelvam Venkatesh , Jiayin Shen , Karson S. Putt , Philip S. Low 3,4
1,2
3
4
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.
Correspondence to: Dr. Philip S. Low, Department of Chemistry, Purdue University, 720 Clinic Drive, West Lafayette, IN 47907,
USA. E-mail: plow@purdue.edu

How to cite this article: Venkatesh C, Shen J, Putt KS, Low PS. Evaluation of the reducing potential of PSMA-containing endosomes
by FRET imaging. Cancer Drug Resist 2021;4:223-32. http://dx.doi.org/10.20517/cdr.2020.84

Received: 16 Sep 2020 First Decision: 9 Oct 2020 Revised: 16 Oct 2020 Accepted: 2 Nov 2020 Available online: 19 Mar 2021
Academic Editor: Godefridus J. Peters Copy Editor: Cai-Hong Wang Production Editor: Jing Yu

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.

© The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0
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sharing, adaptation, distribution and reproduction in any medium or format, for any purpose, even commercially, as long
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