Disulfiram Is a Potent Inhibitor of Proteases of the Caspase Family

Abstract

We have recently shown that dithiocarbamate (DC) disulfides inhibit proteolytic processing of the caspase-3 proenzyme in Jurkat T lymphocytes treated with anti-CD95 (Fas/APO-1) antibody. Because the processing can be accomplished by caspase activity, we investigated the effect of DC disulfides, such as disulfiram (DSF), on active caspases. DSF showed a dose-dependent inhibition was prevented by including dithiothreitol (DTT) in the reaction buffer, thiol-disulfide exchange between inhibitor and target is suggested. Direct interaction of DSF with caspases was confirmed by its inhibition of the purified Ac-DEVD-AMC cleaving protease, caspase-3 (CPP32/apopain). An apparent rate constant (K(app)) for this inhibition was estimated to be 0.45 x 10(3)M(-1)s(-1). DSF was also observed to inhibit the purified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1 beta-converting enzyme, ICE), with a K(app) of 2.2 x 10(3) M(-1)s(-1). In this case protein mixed disulfide formation between DSF and caspase-1 was directly demonstrated using 35S-labeled DSF. The physiological disulfide GSSG was also observed to influence the activity of caspases. A glutathione buffer (5 mM) with a GSH:GSSG ratio of 9:1 decreased the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts by 50% as compared to GSH controls without GSSG. In conclusion, our study shows that caspases are quite sensitive to thiol oxidation and that DSF is a very potent oxidant of caspase protein thiol(s), being 700-fold more potent than glutathione disulfide.

Glutathione S-Transferase Omega 1-1 Is a Target of Cytokine Release Inhibitory Drugs and May Be Responsible for Their Effect on Interleukin-1औ Posttranslational Processing

“…Caspases, as a family of cysteine proteases, represent prime targets for glutathionylation at their active center thiols. Indeed, purified caspase-3 forms a mixed disulfide and is inactivated following treatment with oxidized glutathione (87). Interestingly, activation of caspase-8 via the death-inducing signaling complex in FAS-induced lymphocytes is dependent on the presence of glutathione (88).…”

Antiviral Effects of Pyrrolidine Dithiocarbamate on Human Rhinoviruses

“…Thiuram disulfides are responsible for the prooxidant effects of dithiocarbamates characterized by their potent oxidation of glutathione and protein thiols (48,50). There is evidence that this prooxidative effect inactivates caspases by thiol oxidation and thereby blocks apoptosis (49). As the picornaviral proteinases 2A and 3C are cysteine proteinases, they are highly sensitive to oxidation and are strictly dependent on the reducing environment of the cell.…”

Oxidative stress induces caspase-independent retinal apoptosis in vitro

“…60 It is becoming evident that the cellular reducing environment maintained by endogenous antioxidants such as glutathione and thioredoxin 59 plays an important role in the ability of the caspases to execute the effector stages of the apoptotic program.…”