DISULFIRAM IS A POTENT INHIBITOR OF PROTEASES OF THE CASPASE FAMILY

We have recently shown that dithiocarbamate (DC) disulfides inhibit pr oteolytic processing of the caspase-3 proenzyme in Jurkat T lymphocyte s 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 show ed a dose-dependent inhibition of the Ac-DEVD-AMC cleaving activity in S100 cytosolic extracts prepared from CD95-activated Jurkat cells. Si nce reduced diethyldithiocarbamate had no effect and DSF inhibition wa s prevented by including dithiothreitol (DTT) in the reaction buffer, thiol-disulfide exchange between inhibitor and target is suggested. Di rect interaction of DSF with caspases was confirmed by its inhibition of the purified Ac-DEVD-AMC cleaving protease, caspase-3 (CPP32/apopai n). An apparent rate constant (K-app) for this inhibition was estimate d to be 0.45 x 10(3) M-1 s(-1). DSF was also observed to inhibit the p urified Ac-YVAD-AMC cleaving enzyme, caspase-1 (interleukin-1 beta-con verting enzyme, ICE), with a K-app of 2.2 x 10(3) M-1 s(-1) In this ca se protein mixed disulfide formation between DSF and caspase-1 was dir ectly demonstrated using S-35-labeled DSF. The physiological disulfide GSSG was also observed to influence the activity of caspases. A gluta thione buffer (5 mM) with a GSH:GSSG ratio of 9:1 decreased the Ac-DEV D-AMC cleaving activity in S100 cytosolic extracts by 50% as compared to GSH controls without GSSG. In conclusion, our study shows that casp ases are quite sensitive to thiol oxidation and that DSF is a very pot ent oxidant of caspase protein thiol(s), being 700-fold more potent th an glutathione disulfide.