Thioredoxin reductase, an emerging target for anticancer metallodrugs. Enzyme inhibition by cytotoxic gold(III) compounds studied with combined mass spectrometry and biochemical assays

The seleno-enzyme thioredoxin reductase (TrxR) is a putative target for cytotoxic gold complexes. We
investigated the mechanism of TrxR inhibition by a group of structurally diverse gold(III) compounds;
the antiarthritic gold(I) drugs auranofin and aurothiomalate were also studied for comparison
purposes. The tested compounds – either gold(III) or gold(I) – were found to produce potent enzyme
inhibition only after pre-reduction of the enzyme with NADPH, indicating that TrxR inhibition is the
result of protein structure modifications occurring upon cofactor binding. MALDI-ToF MS
experiments on the intact enzyme provided evidence for extensive enzyme metallation, while
experiments on trypsinized gold(III)-protein adducts identified a specific protein fragment – namely
236IGEHMEEHGIK246 – bearing an attached gold(I) ion. Independent mechanistic information on the
system was derived from BIAM assays capable of monitoring selective metal binding to cysteine and/or
selenocysteine residues. While the effects produced by auranofin could be essentially ascribed to gold(I)
coordination to the active site selenol, the effects caused by the various gold(III) compounds were better
interpreted in terms of oxidative protein damage.