Pronounced toxicity differences between homobifunctional protein cross-linkers and analogous monofunctional electrophiles.

Bifunctional electrophiles have been used in various chemopreventive, chemotherapeutic, and bioconjugate applications. Many of their effects in biological systems are traceable to their reactive properties, whereby they can modify nucleophilic sites in DNA, proteins, and other cellular molecules. Previously, we found that two different bifunctional electrophiles--diethyl acetylenedicarboxylate and divinyl sulfone--exhibited a strong enhancement of toxicity when compared with analogous monofunctional electrophiles in both human colorectal carcinoma cells and baker's yeast. Here, we have compared the toxicities for a broader panel of homobifunctional electrophiles bearing diverse electrophilic centers (e.g., isothiocyanate, isocyanate, epoxide, nitrogen mustard, and aldehyde groups) to their monofunctional analogues. Each bifunctional electrophile showed at least a 3-fold enhancement of toxicity over its monofunctional counterpart, although in most cases, the differences were even more pronounced. To explain their enhanced toxicity, we tested the ability of each bifunctional electrophile to cross-link recombinant yeast thioredoxin 2 (Trx2), a known intracellular target of electrophiles. The bifunctional electrophiles were capable of cross-linking Trx2 to itself in vitro and to other proteins in cells exposed to toxic concentrations. Moreover, most cross-linkers were preferentially reactive with thiols in these experiments. Collectively, our results indicate that thiol-reactive protein cross-linkers in general are much more potent cytotoxins than analogous monofunctional electrophiles, irrespective of the electrophilic group studied.

Bifunctional electrophiles cross-link thioredoxins with redox relay partners in cells.

Thioredoxin protects cells against oxidative damage by reducing disulfide bonds in improperly oxidized proteins. Previously, we found that the baker's yeast cytosolic thioredoxin Trx2 undergoes cross-linking to form several protein-protein complexes in cells treated with the bifunctional electrophile divinyl sulfone (DVSF). Here, we report that the peroxiredoxin Tsa1 and the thioredoxin reductase Trr1, both of which function in a redox relay network with thioredoxin, become cross-linked in complexes with Trx2 upon DVSF treatment. Treatment of yeast with other bifunctional electrophiles, including diethyl acetylenedicarboxylate (DAD), mechlorethamine (HN2), and 1,2,3,4-diepoxybutane (DEB), resulted in the formation of similar cross-linked complexes. Cross-linking of Trx2 and Tsa1 to other proteins by DVSF and DAD is dependent on modification of the active site Cys residues within these proteins. In addition, the human cytosolic thioredoxin, cytosolic thioredoxin reductase, and peroxiredoxin 2 form cross-linked complexes to other proteins in the presence of DVSF, although each protein shows different susceptibilities to modification by DAD, HN2, and DEB. Taken together, our results indicate that bifunctional electrophiles potentially disrupt redox homeostasis in yeast and human cells by forming cross-linked complexes between thioredoxins and their redox partners.