Unlocking the Power of Human Ferritin: Enhanced Drug Delivery of Aurothiomalate in A2780 Ovarian Cancer Cells.

Aurothiomalate (AuTM) is an FDA-approved antiarthritic gold drug with unique anticancer properties. To enhance its anticancer activity, we prepared a bioconjugate with human apoferritin (HuHf) by attaching some AuTM moieties to surface protein residues. The reaction of apoferritin with excess AuTM yielded a single adduct, that was characterized by ESI MS and ICP-OES analysis, using three mutant ferritins and trypsinization experiments. The adduct contains ~3 gold atoms per ferritin subunit, arranged in a small cluster bound to Cys90 and Cys102. MD simulations provided a plausible structural model for the cluster. The adduct was evaluated for its pharmacological properties and was found to be significantly more cytotoxic than free AuTM against A2780 cancer cells mainly due to higher gold uptake. NMR-metabolomics showed that AuTM bound to HuHf and free AuTM induced qualitatively similar changes in treated cancer cells, indicating that the effects on cell metabolism are approximately the same, in agreement with independent biochemical experiments. In conclusion, we have demonstrated here that a molecularly precise bioconjugate formed between AuTM and HuHf exhibits anticancer properties far superior to the free drug, while retaining its key mechanistic features. Evidence is provided that human ferritin can serve as an excellent carrier for this metallodrug.

The Many Lives of Auranofin: How an Old Anti-Rheumatic Agent May Become a Promising Antimicrobial Drug.

Auranofin (AF) is a gold-based compound with a well-known pharmacological and toxicological profile, currently used in the treatment of some severe forms of rheumatoid arthritis. Over the last twenty years, AF has also been repurposed as antiviral, antitumor, and antibacterial drug. In this review we focused on the antibacterial properties of AF, specifically researching the minimal inhibitory concentrations (MIC) of AF in both mono- and diderm bacteria reported so far in literature. AF proves to be highly effective against monoderm bacteria, while diderm are far less susceptible, probably due to the outer membrane barrier. We also reported the current mechanistic hypotheses concerning the antimicrobial properties of AF, although a conclusive description of its antibacterial mode of action is not yet available. Even if its mechanism of action has not been fully elucidated yet and further studies are required to optimize its delivery strategy, AF deserves additional investigation because of its unique mode of action and high efficacy against a wide range of pathogens, which could lead to potential applications in fighting antimicrobial resistance and improving therapeutic outcomes in infectious diseases.

Mercury binding to proteins disclosed by ESI MS experiments: The case of three organomercurials.

Solution interactions of three organomercury compounds, i.e., methylmercury chloride, thimerosal and phenylmercury acetate, with a group of biochemically relevant proteins, namely cytochrome c (Cyt c), ribonuclease A (RNase A), carbonic anhydrase I (hCA I), superoxide dismutase (SOD), and serum albumin (HSA), were investigated using an established ESI MS approach. Temporal analysis of sample aliquots provided insight into the binding kinetics, while comparative analysis of the obtained mass spectra disclosed adduct formation of each mercurial with the tested proteins and the relative abundance of the species. The three organomercurials bind, exclusively and tightly, to free cysteine residues as no binding was observed in the case of proteins lacking such groups. hCA I, SOD and HSA formed distinct mercury adducts, preserving the Hg bound alkyl/aryl ligands; yet, the three organomercurials displayed significant differences in reactivity in relation to their chemical structure. The investigation was then extended to analyze the reactions with the C-terminal dodecapeptide of the enzyme human thioredoxin reductase, which contains a characteristic selenol-thiol moiety: tight Hg binding was observed. Notably, this peptide was able to remove effectively and completely the alkyl/aryl ligands of the three tested organomercurials; this behavior may be relevant to the detoxification mechanism of organomercurials in mammals. Finally, a competition experiment was carried out to establish whether protein bound mercury centers may be displaced by other competing metals. Interestingly, and quite unexpectedly, we observed that a protein bound mercury fragment may be partially displaced from its coordination site in hCA I by the medicinal gold compound auranofin.

The effects of two cytotoxic gold(i) carbene compounds on the metabolism of A2780 ovarian cancer cells: mechanistic inferences through NMR analysis.

NMR metabolomics is a powerful tool to characterise the changes in cancer cell metabolism elicited by anticancer drugs. Here, the large metabolic alterations produced by two cytotoxic gold carbene compounds in A2780 ovarian cancer cells are described and discussed in comparison to auranofin, in the frame of the available mechanistic knowledge.

19F: A small probe for a giant protein.

Herein we describe a method for the efficient production (∼90% fluorination) of 5-F-Trp human H ferritin via the selective incorporation of 19F into the side chain of W93 using 5-fluoroindole as the fluorinated precursor of the amino acid. Human H ferritin is a nanocage composed of 24 identical subunits, each containing a single Trp belonging to a loop exposed on the external surface of the protein nanocage. This makes 5-F-Trp a potential probe for the study of intermolecular interactions in solution by exploiting its intrinsic fluorescence. More interestingly, albeit the large size of the cage (12 nm external diameter, ∼500 kDa molecular mass) we observe a broad but well defined NMR 19F resonance that can be used for the dual purpose of detecting solution intermolecular interactions via chemical shift perturbation mapping and monitoring the uptake of ferritin by cells treated with ferritin-based drug carriers, the latter being an application area of increasing importance.