The study of the mode-of-action of Auranofin, a cytotoxic gold(i) compound, reveals that it binds exclusively to the free and solvent-accessible cysteine residues of biologically relevant proteins.

Electrospray ionization mass spectrometry (ESI MS) is a powerful investigative tool to analyze the reactions of metallodrugs with proteins and peptides and characterize the resulting adducts. Here, we have applied this type of approach to four experimental anticancer gold(III) compounds for which extensive biological and mechanistic data had previously been gathered, namely, Auoxo6, Au 2 phen, AuL12, and Aubipyc. These gold(III) compounds were reacted with two representative proteins, i.e., human serum albumin (HSA) and human carbonic anhydrase I (hCA I), and with the C-terminal dodecapeptide of thioredoxin reductase. ESI MS analysis allowed us to elucidate the nature of the resulting metal-protein adducts from which the main features of the occurring metallodrug-protein reactions can be inferred. In selected cases, MS data were integrated and supported by independent 1 HNMR and UV-Vis absorption measurements to gain an overall description of the occurring processes. From data analysis, it emerges that most of the investigated gold(III) complexes, endowed with an appreciable oxidizing character, undergo quite facile reduction to gold(I); the resulting gold(I) species tightly associate with the above proteins/peptides with a remarkable selectivity for free cysteine residues. In contrast, in the case of the less-oxidizing Aubipyc complex, the gold(III) oxidation state is conserved, and a gold(III) fragment still containing the original ligand is found to be associated with the target proteins. It is notable that the C-terminal dodecapeptide of thioredoxin reductase containing the characteristic-Gly-Cys-Sec-Gly metal-binding motif is able in all cases to trigger gold(III)-to-gold(I) reduction. Our investigation allowed us to identify in detail the nature of the gold fragments that ultimately bind the protein targets and determine the exact binding stoichiometry; some insight on the reaction kinetics was also gained. Notably, a few clear correlations could be established between the structure of the metal complexes and the nature of the resulting protein adducts. The mechanistic implications of these findings are analyzed and thoroughly discussed. Overall, the present results set the stage to better understand the real target biomolecules of these gold compounds and elucidate at the atomic level their interaction modes with proteins and peptides.

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Native mass spectrometry is a potent technique to study and characterize biomacromolecules in their native state. Here, we have applied this method to explore the solution chemistry of human carbonic anhydrase I (hCA I) and its interactions with four different inhibitors, namely three sulfonamide inhibitors (AAZ, MZA, SLC-0111) and the dithiocarbamate derivative of morpholine (DTC). Through high-resolution ESI-Q-TOF measurements, the native state of hCA I and the binding of the above inhibitors were characterized in the molecular detail. Native mass spectrometry was also exploited to assess the direct competition in solution among the various inhibitors in relation to their affinity constants. Additional studies were conducted on the interaction of hCA I with the metallodrug auranofin, under various solution and instrumental conditions. Auranofin is a selective reagent for solvent-accessible free cysteine residues, and its reactivity was analyzed also in the presence of CA inhibitors. Overall, our investigation reveals that native mass spectrometry represents an excellent tool to characterize the solution behavior of carbonic anhydrase. Graphic abstract