Suppression of Human T Cell Proliferation Mediated by the Cathepsin B Inhibitor, z-FA-FMK Is Due to Oxidative Stress.

Tanuja Rajah,Sek Chuen Chow

doi:10.1371/journal.pone.0123711

Abstract

The cathepsin B inhibitor, benzyloxycarbonyl-phenylalanine-alanine-fluoromethyl ketone (z-FA-FMK) readily inhibits anti-CD3-induced human T cell proliferation, whereas the analogue benzyloxycarbonyl-phenylalanine-alanine-diazomethyl ketone (z-FA-DMK) had no effect. In contrast, benzyloxycarbonyl-phenylalanine-alanine-chloromethyl ketone (z-FA-CMK) was toxic. The inhibition of T cell proliferation mediated by z-FA-FMK requires not only the FMK moiety, but also the benzyloxycarbonyl group at the N-terminal, suggesting some degree of specificity in z-FA-FMK-induced inhibition of primary T cell proliferation. We showed that z-FA-FMK treatment leads to a decrease in intracellular glutathione (GSH) with a concomitant increase in reactive oxygen species (ROS) levels in activated T cells. The inhibition of anti-CD3-induced T cell proliferation mediated by z-FA-FMK was abolished by the presence of low molecular weight thiols such as GSH, N-acetylcysteine (NAC) and L-cysteine, whereas D-cysteine which cannot be metabolised to GSH has no effect. The inhibition of anti-CD3-induced up-regulation of CD25 and CD69 expression mediated by z-FA-FMK was also attenuated in the presence of exogenous GSH. Similar to cell proliferation, GSH, NAC and L-cysteine but not D-cysteine, completely restored the processing of caspase-8 and caspase-3 to their respective subunits in z-FA-FMK-treated activated T cells. Our collective results demonstrated that the inhibition of T cell activation and proliferation mediated by z-FA-FMK is due to oxidative stress via the depletion of GSH.

Highlights

Halomethylketone peptides such as peptidyl chloromethylketones were the first active site directed irreversible enzyme inhibitors synthesised and were originally designed as potential drugs for the treatment of certain diseases [1,2]
We examined whether the benzyloxycarbonyl (z) group at the N-terminal of zFA-FMK play any role in blocking T cell activation and proliferation
These results suggest that the benzyloxycarbonyl group at the N-terminal of the peptidyl methylketone plays a role in the immunosuppressive effects of z-FA-FMK

Summary

Introduction

Halomethylketone peptides such as peptidyl chloromethylketones were the first active site directed irreversible enzyme inhibitors synthesised and were originally designed as potential drugs for the treatment of certain diseases [1,2]. The highly electrophilic chloromethylketone moiety was too reactive and results in the alkylation of non-target molecules indiscriminately [3,4]. Efforts to replace the reactive chlorine atom led to the eventual synthesis of peptidyl fluoromethylketones [3]. April 27, 2015 z-FA-FMK Blocks T Cell Proliferation via Oxidative Stress relative to carbon-chlorine bonds, fluoromethylketones were expected to be poorer alkylating agents and should reduce the non-specific alkylation significantly compared to chloromethylketones. Once synthesised, peptidyl fluoromethylketones were found to be highly reactive and are selective irreversible inhibitors for cysteine proteases [4]

Methods

Results

Conclusion