MECHANISMS OF INHIBITION OF THE THIOREDOXIN GROWTH-FACTOR SYSTEM BY ANTITUMOR 2-IMIDAZOLYL DISULFIDES

The interactions of a series of 2-imidazolyl disulfide antitumor compo unds with the thioredoxin reductase(TR)/thioredoxin (hTrx) redox syste m have been studied. Disulfides III-2 (n-butyl 2-mercaptoimidazolyl di sulfide) and VI-2 (ethyl 2-mercaptoimidazolyl disulfide) were substrat es for reduction by TR with K-m values of 43 and 48 mu M. Disulfides I V-2 (1-methylpropyl 2-mercaptoimidazolyl disulfide) and DLK-36 (benzyl 2-mercaptoimidazolyl disulfide) were competitive inhibitors of the re duction of hTrx by TR with K-i values of 31 mu M. None of the disulfid es were substrates for reduction by human glutathione reductase. The d isulfides caused reversible thioalkylation of hTrx at the redox cataly tic site as shown by the fact that there was no thioalkylation of a mu tant hTrx where both the catalytic site Cys(32) and Cys(35) residues w ere replaced by Ser. In addition, the disulfides caused a slower irrev ersible inactivation of hTrx as a substrate for reduction by TR, with half-lives for III-2 of 30 min, for IV-2 of 4 hr, and for IX-2 (t-buty l 2-mercaptoimidazolyl disulfide) of 24 hr. This irreversible inactiva tion of hTrx occurred at concentrations of the disulfides an order of magnitude below those that inhibited TR, and involved the Cys(73) of h Trx, which is outside the conserved redox catalytic site, as shown by the resistance to inactivation of a mutant hTrx where Cys(73) was repl aced by Ser. Electrophoretic and mass spectral analyses of the product s of the reaction between the disulfides and hTrx show that modificati on of 1-3 Cys residues of the protein occurred in a concentration depe ndent fashion. The disulfides inhibited the hTrx-dependent proliferati on of MCF-7 breast cancer cells with IC50 values for III-2 and IV-2 of 0.2 and 1.2 mu M, respectively. The results show that although the ca talytic sites of TR and hTrx are reversibly inhibited by the 2-imidazo lyl disulfides, it is the irreversible thioalkylation of Cys(73) of hT rx by the disulfides that most probably accounts for the inhibition of thioredoxin-dependent cell growth by the disulfides. (C) 1998 Elsevie r Science Inc.