Toxicity of the peptidyl halomethylketone z-FA-CMK in T cells

Abstract

Peptidyl halomethylketones are synthetic proteas inhibitors designed to block the activity of cysteine or serine proteases. Recently, the cathepsin inhibitor, benzyloxycarbonyl-phe-ala-fluoromethylketone (z-FA-FMK) with alanine and phenylalanine at the P1 and P2 positions, respectively, has been shown to have immunosuppressive properties in vitro and in vivo. Structure-activity relationship studies on z-FA-FMK revealed that replacing the fluorine atom with chlorine at the methylketone moiety renders the chloromethylketone (CMK) analogue, z-FA-CMK very toxic to Jurkat T cells. Due to the highly reactive CMK moiety, peptidyl CMKs are known to be potentially toxic. However, the present study showed that peptide derived with a CMK moiety is not necessary toxic to Jurkat T cells and that almost all the structural components is crucial in z-FA-CMK toxicity, suggesting that the toxicity mediated by z-FA-CMK is rather specific. Dose-dependent studies revealed that lower concentrations of z-FA-CMK induced apoptosis in Jurkat T cells whereas higher concentrations induced necrosis. Apoptosis but not necrosis induced by z-FA-CMK is readily blocked by caspase inhibitors. However, both apoptosis and necrosis were blocked by the antioxidant N-acetylcysteine, suggesting that oxidative stress may be involved in the mechanism of z-FA-CMK toxicity. Further studies showed that toxicity of z-FA-CMK in T cells involves the depletion of intracellular glutathione, generation of reactive oxygen species, and the collapse of the mitochondrial membrane potential. As predicted for peptidyl CMK, z-FA-CMK depleted glutathione, an effect which can cause ROS generation and oxidative stress. However, results in the present study suggested that the generation of ROS plays a more important role than glutathione depletion in the mechanism of oxidative stress induced by z-FA-CMK toxicity. In conclusion, the peptidyl halomethylketone z-FA-CMK was found to be highly toxic in the leukemic T cell line, Jurkat and exert its toxicity via oxidative stress. Compared with primary T cells (mouse thymocytes), Jurkat T cells appeared to be more susceptible to z-FA-CMK toxicity. However, this remained to be furthered investigated.