Reactions of Medicinal Gold(III) Compounds With Proteins and Peptides Explored by Electrospray Ionization Mass Spectrometry and Complementary Biophysical Methods

Abstract

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.

Anticancer Diiron Vinyliminium Complexes: A Structure–Activity Relationship Study

“…During the past years, high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) has emerged as a valuable and powerful tool to study the formation of adducts derived from the interaction of metallodrugs with biomolecules [ 76 , 77 , 78 ]. Moreover, this technique allows the identification of the precise nature of the fragments attached to protein side chains and, thus, to infer some relevant mechanistic information [ 79 , 80 , 81 ].…”

A C∧S-Cyclometallated Gold(III) Complex as a Novel Antibacterial Candidate Against Drug-Resistant Bacteria

“…Both mechanisms may lead to bacterial cell death. The nucleophilic displacement of bromide in gold(III) complexes by free cysteine residues of proteins has been established by electrospray ionization – mass spectrometry (ESI-MS) studies ( Massai et al, 2020 ).…”

Thermodynamic Evaluation of the Interactions between Anticancer Pt(II) Complexes and Model Proteins

“…The reactivity behaviour of complexes 1 – 3 towards the two model proteins was also evaluated using high-resolution ESI mass spectrometry. For these experiments, the proteins were treated in physiological-like conditions, using a well-established protocol [ 18 , 36 , 37 ] that can assure a protein reactivity as similar as possible to that of the physiological environment. Moreover, the spectra were acquired with a methodology able to preserve the protein in its native state, conserving unaltered all the covalent interactions formed in solution and most of the non-covalent ones [ 38 ].…”

“…The consensus on the crucial role of the binding of metallodrugs with protein targets has grown [ 14 , 15 ]. Some of us were already involved in the study of metallodrugs interaction with small molecular models mimicking the protein coordinative sites [ 16 , 17 ] and with proteins of bigger size [ 18 , 19 , 20 ].…”