Improved Cathepsin Probes for Sensitive Molecular Imaging.

Yonit Yitzhak,Hanmant Gaikwad,Boris Turk,Galia Blum,Emmanuelle Merquiol,Tommy Weiss-Sadan

doi:10.3390/molecules27030842

Abstract

Cysteine cathepsin proteases are found under normal conditions in the lysosomal compartments of cells, where they play pivotal roles in a variety of cellular processes such as protein and lipid metabolism, autophagy, antigen presentation, and cell growth and proliferation. As a consequence, aberrant localization and activity contribute to several pathologic conditions such as a variety of malignancies, cardiovascular diseases, osteoporosis, and other diseases. Hence, there is a resurgence of interest to expand the toolkit to monitor intracellular cathepsin activity and better ascertain their functions under these circumstances. Previous fluorescent activity-based probes (ABPs) that target cathepsins B, L, and S enabled detection of their activity in intact cells as well as non-invasive detection in animal disease models. However, their binding potency is suboptimal compared to the cathepsin inhibitor on which they were based, as the P1 positive charge was capped by a reporter tag. Here, we show the development of an improved cathepsin ABP that has a P1 positive charge by linking the tag on an additional amino acid at the end of the probe. While enhancing potency towards recombinant cathepsins, the new probe had reduced cell permeability due to additional peptide bonds. At a second phase, the probe was trimmed; the fluorophore was linked to an extended carbobenzoxy moiety, leading to enhanced cell permeability and superb detection of cathepsin activity in intact cells. In conclusion, this work introduces a prototype design for the next generation of highly sensitive ABPs that have excellent detection of cellular cathepsin activity.

Highlights

Cysteine cathepsins (CTSs) are proteolytic enzymes that are involved in a variety of physiologic processes
In an attempt to improve the potency of our published cathepsin activity-based probes (ABPs), GB123, we generated a probe that has a positive charge at the P1 position
To increase cell permeability, the Cy5 fluorophore of GB123 was replaced with a BODIPY TMR-X fluorophore, Scheme 1

Summary

Introduction

Cysteine cathepsins (CTSs) are proteolytic enzymes that are involved in a variety of physiologic processes These include housekeeping activities such as autophagy and transcriptional control, but numerous pieces of evidence implicate them in different pathologic conditions such as inflammatory diseases and cancer [1,2,3]. Members of the cathepsin family, CTS B, L, and S, were believed to require acidic conditions such as in the lysosomes [2,3,4] This view was refuted, as CTSs were evident in unexpected cellular compartments such as the cytosol, nucleus, and extracellular milieu [1,4,5,6,7]. These, are hindered due to the lack of sensitive tools to monitor CTS activity Such activities include nuclear cathepsin function during the cell cycle or under conditions of cellular starvation. Tools that report on activity are needed to investigate CTS involvement in these activities

Methods

Results

Conclusion