Thioredoxin reductase, an emerging target for anticancer metallodrugs. Enzyme inhibition by cytotoxic gold(iii) compounds studied with combined mass spectrometry and biochemical assaysElectronic supplementary information (ESI) available: Experimental section, Table S1, Figure S1, S2 and S3. See DOI: 10.1039/c0md00181c

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 236 IGEHMEEHGIK 246 - 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. The mechanism of thioredoxin reductase inhibition by cytotoxic gold( iii ) compounds is studied by a combined approach.