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Photoactivatable Pt(IV) prodrugs represent nowadays an intriguing class of potential metal-based drugs, endowed with more chemical inertness in their oxidized form and better selectivity for the target with respect to the clinically established Pt(II) compounds. In fact, they have the possibility to be reduced by light irradiation directly at the site of interest. For this reason, we synthesized a new Pt(IV) complex, [Pt(OCOCH3)3(4′-phenyl-2,2′:6′,2′′-terpyridine)][CF3SO3] (1), that is well soluble in aqueous medium and totally unreactive towards selected model biomolecules until its reduction. The highlight of this work is the rapid and efficient photoreduction of 1 with visible light (460 nm), which leads to its reactive Pt(II) analogue. This behavior was made possible by taking advantage of an efficient catalytic system based on flavin and NADH, which is naturally present in the cellular environment. As a comparison, the reduction of 1 was also studied with simple UV irradiation, but both UV-Vis spectrophotometry and 1H-NMR spectrometry showed that the flavin-catalyzed reduction with visible light was faster. Lastly, the reactivity against two representative biological targets, i.e., human serum albumin and one monofilament oligonucleotide fragment, was evaluated by high-resolution mass spectrometry. The results clearly pointed out that the prodrug 1 did not interact with these targets until its photoreduction to the Pt(II) analogue.

Although important progress has been made, cancer still remains a complex disease to treat. Serious side effects, the insurgence of resistance and poor selectivity are some of the problems associated with the classical metal-based anti-cancer therapies currently in clinical use. New treatment approaches are still needed to increase cancer patient survival without cancer recurrence. Herein, we reviewed two promising—at least in our opinion—new strategies to increase the efficacy of transition metal-based complexes. First, we considered the possibility of assembling two biologically active fragments containing different metal centres into the same molecule, thus obtaining a heterobimetallic complex. A critical comparison with the monometallic counterparts was done. The reviewed literature has been divided into two groups: the case of platinum; the case of gold. Secondly, the conjugation of metal-based complexes to a targeting moiety was discussed. Particularly, we highlighted some interesting examples of compounds targeting cancer cell organelles according to a third-order targeting approach, and complexes targeting the whole cancer cell, according to a second-order targeting strategy.

AuIII complexes with N‐heterocyclic carbene (NHC) ligands have shown remarkable potential as anticancer agents, yet their fate in vivo has not been thoroughly examined and understood. Reported herein is the synthesis of new AuIII‐NHC complexes by direct oxidation with radioactive [124I]I2 as a valuable strategy to monitor the in vivo biodistribution of this class of compounds using positron emission tomography (PET). While in vitro analyses provide direct evidence for the importance of AuIII‐to‐AuI reduction to achieve full anticancer activity, in vivo studies reveal that a fraction of the AuIII‐NHC prodrug is not immediately reduced after administration but able to reach the major organs before metabolic activation., Images: The reaction of [124/131I]I2 with AuI‐N‐heterocyclic carbene precursors affords a strategy to label AuIII anticancer agents and study their biodistribution in vivo by positron emission tomography.

The use of Pt(iv) complexes as potential anticancer drugs is attractive, because they have higher stability and less side effects than Pt(ii) compounds. Moreover, some Pt(iv) complexes can also be activated with light, opening an avenue to photochemotherapy. Our purpose is to widen the library of photoactivatable Pt(iv)-based prodrugs and here we report on the oxidation of the Pt(ii) compound [PtCl(4'-phenyl-2,2':6',2''-terpyridine)][CF3SO3] (1) with PhICl2 or H2O2. The synthetic procedure avoids the formation of multiple species: the treatment with PhICl2 produces the Pt(iv) complex with axial chlorides, [PtCl3(4'-phenyl-2,2':6',2''-terpyridine)][CF3SO3] (2), while H2O2 oxidation and post-synthesis carboxylation produce [Pt(OCOCH3)2Cl(4'-phenyl-2,2':6',2''-terpyridine)][CF3SO3] (3), bearing acetates in the axial positions. 2 and 3 are stable in physiological-like buffers and in DMSO in the dark, but undergo photoreduction to 1 upon irradiation at 365 nm. Their stability toward reduction is a fundamental parameter to consider: cyclic voltammetry experiments show that the 2 electron reduction Pt(iv) → Pt(ii) occurs at a more negative potential for 3, because of the greater stabilization provided by the acetate axial groups; noteworthily, 3 is stable for hours also in the presence of mM concentration of glutathione. The cytotoxicity of 2 and 3 toward A2780 and A2780cis cell lines reveals that 3 is the least toxic in the dark, but is able to produce cytotoxic effects far higher than cisplatin when irradiated. To shed light on the mechanistic aspects, the interaction with protein and DNA models has been explored through high-resolution mass spectrometry revealing that 2 and 3 behave as prodrugs, but are able to bind to biological targets only after irradiation.

Despite the widespread use of platinum drugs in the treatment of colorectal cancer (CRC), due to the heavy side effects and to intrinsic or acquired Pt resistance, new and more efficient drugs are urgently needed. Starting from the encouraging results obtained for the complex PtI2(DACH), we summarise here our recent advances, reporting data on the synthesis and the chemical and biological features of two oxaliplatin analogues i.e. PtBr2(DACH) and PtCl2(DACH). The comparative approach of these studies reveals how these analogues possess interesting and differential pharmacological properties as well as some peculiar features that may be conveniently exploited to shed light in the mechanistic aspects involved in the pharmacological action of the parent drug oxaliplatin. Furthermore, these findings may inspire the design of more effective Pt-based anticancer drugs to be used in CRC treatment.

The solution behavior of auranofin, Et3PAuCl and Et3PAuI, as well as their interactions with hen egg white lysozyme, single strand oligonucleotide, and ds-DNA were comparatively analyzed through NMR spectroscopy, ESI-MS, ethidium bromide displacement, DNA melting and viscometric tests. The cytotoxic effects toward representative colorectal cancer cell lines were found to be strong and similar in the three cases and a good correlation could be established between the cytotoxicity and the ability to inhibit thioredoxin reductase; remarkably, in vivo acute toxicity experiments for Et3PAuI confirmed that, similarly to auranofin, this drug is well tolerated in a murine model. Overall, a very similar profile emerges for Et3PAuI and Et3PAuCl, which retain the potent cytotoxic effects of auranofin while showing some peculiar features. These results demonstrate that the presence of the thiosugar moiety is not mandatory for the pharmacological action, suggesting that the tuning of some relevant chemical properties...

Au(III) complexes with N-Heterocyclic Carbenes (NHCs) ligands have shown remarkable potential as anticancer agents, yet their fate in vivo has not been thoroughly examined and understood. Herein we report on the synthesis of new Au(III)-NHC complexes via direct oxidation with radioactive [124I]I2 as a valuable strategy to monitor the in vivo biodistribution of this class of compounds using positron emission tomography (PET) and, in combination with in vitro analyses, to provide direct evidence of the importance of Au(III)-to-Au(I) reduction for achieving full anticancer activity.

A silver(I) and a gold(I) complex of the fluorescent N-heterocyclic carbenic (NHC) ligand 1-(9-anthracenylmethyl)-3-(3-trimethylsilyl-2-propynil)-benzimidazol-2-ylidene have been synthesized and characterized. These compounds show cytotoxicity in the micromolar range and higher antiproliferative properties than cisplatin (CDDP) against several tumour cell lines such as SW480 (colon), A549 (lung) and HepG2 (liver). Both metal complexes are successfully internalized by SW480 cells being the silver compound the most accumulated. Subsequently, they were evaluated as inhibitors of the selenoenzyme Thioredoxin reductase (TrxR) and as DNA binders. Fluorescence microscopy confirmed that both protein and DNA binding could be involved in the biological activity of the compounds. The silver carbene was the most effective enzyme inhibitor with an IC50 in the nanomolar range. Also, interaction studies with natural double stranded DNA highlight a strong stabilisation of the double helix after binding to the Ag(I) carbene, indicating its potential suitability as dual-targeting anticancer active molecule., “la Caixa” Foundation (LCF/PR/PR12/11070003), Ministerio de Ciencia Innovación y Universidades-FEDER (RTI2018-102040-B-100) and Junta de Castilla y León-FEDER (BU305P18).

The reactions with a few model proteins of two structurally related gold carbene compounds, namely the gold(I) monocarbene complex Au(NHC)Cl and the corresponding bis-carbene complex [Au(NHC)2]PF6 (where NHC is an N-heterocyclic carbene ligand), were comparatively studied by ESI MS measurements. The investigated proteins were: human serum albumin, human carbonic anhydrase and bovine superoxide dismutase; in addition, the reactions of the two gold carbenes with the C-terminal synthetic dodecapeptide of thioredoxin reductase were analyzed. Formation of metallodrug-protein adducts was observed in all cases made exception for the reactions of [Au(NHC)2]PF6 with carbonic anhydrase and superoxide dismutase. Notably, in line with expectations, the monocarbene gold complex turned out to be more effective than its dicarbene counterpart in forming protein adducts. The reactivity of these gold carbene complexes with model proteins is compared to that of a few other gold(III) and gold(I) complexes whose reactions with model proteins had been previously investigated with the same methodology; it emerges that the two gold carbenes react more selectively with proteins at well-defined anchoring sites. The implications of these results are discussed in the light of the current knowledge on medicinal gold compounds.

Metal complexes of N-heterocyclic carbene (NHC) ligands are the object of increasing attention for therapeutic purposes. Among the different metal centres, interest on Au-based compounds started with the application as anti-arthritis drugs. On the other hand, Ag(I) antimicrobial properties have been known for a long time. For Au(I)/Au(III)-NHC and Ag(I)-NHC anti-tumour and anti-proliferative properties have been quite recently demonstrated. In addition to these and as for Group 11, copper is a much less investigated metal centre, but a few papers underline its pharmacological potential. This review wants to focus on the different biological targets for these metal-based compounds. It is divided into chapters which are respectively devoted on: i) mitochondria and thiol oxidoreductase systems; ii) other relevant enzymes; iii) nucleic acids. Examples of representative coinage NHCs for each of the targets are provided together with significant references on recent advances on the topic. Moreover, a final comment summarises the aspects enlightened by each chapter and provides some hints to better understand the metal-NHCs mechanistic behaviour based on structure-activity relationships.

Colorectal cancer (CRC) is a global health problem being the fourth most common cause of death due to cancer worldwide. Oxaliplatin plays a key role in current CRC treatment but shows serious drawbacks, such as a high systemic toxicity and the frequent insurgence of Pt resistance. In search of novel and more efficacious Pt-based drugs for CRC treatment, we synthesized and characterised PtI2(DACH), an oxaliplatin analogue. PtI2(DACH) was obtained through the replacement of bidentate oxalate with two iodides. PtI2(DACH) turned out to be more lipophilic than oxaliplatin, a fact that led to an enhancement of its cellular uptake. In contrast to oxaliplatin, PtI2(DACH) showed a scarce reactivity towards model proteins, while maintaining affinity for a standard DNA oligo. Notably, PtI2(DACH) induced cytotoxicities roughly comparable to those of oxaliplatin in three representative CRC cell lines. Moreover, it was able to trigger cell apoptosis, to an extent even better than cisplatin and oxaliplatin. Overall, a rather promising picture emerges for this novel Pt drug that merits, in our opinion, a deeper and more extensive preclinical evaluation.

The ruthenium-arene complexes [(η6-p-cymene)RuCl2(κP-PPh2(4-C6H4CO2H))], 1, [(η6-p-cymene)RuCl(κ2P,O-PPh2(2-C6H4CO2))], 2, [(η6-p-cymene)RuCl2(κP-PPh2(2-C6H4OCO-EA))], 3, and [(η6-p-cymene)RuCl2(κP-PPh2(4-C6H4CO2CH2CH2OCO-EA))], 4 (EA-CO2H = ethacrynic acid), were synthesized in good to high yields and characterized by analytical techniques, IR, UV-Vis and multinuclear NMR spectroscopy, and single crystal X-ray diffraction in the cases of 1 and 2. The unstable compounds [(η6-arene)RuCl2(κP-PPh2(2-C6H4CO2CH2CH2OCO-EA))] (arene = p-cymene, 5a; arene = C6H6, 5b) were obtained and identified in solution by NMR. Electrochemical and spectro-electrochemical experiments were performed in order to assess the redox behaviour of 1-4 in CH2Cl2. The in vitro cytotoxicity of 1-4 was determined on the human pancreatic cancer cell line BxPC3 and the mouse embryo fibroblast Balb/3T3 Clone A31 cell line, the latter acting as a model for normal cells. Furthermore, the interaction of 1, 3 and 4 with two model proteins was investigated by high resolution ESI-MS.

The trinuclear platinum cluster [Pt3(μ-PBut2)3(CO)3]CF3SO3 (I) was designed featuring the presence of a nearly equilateral platinum triangle bridged by three di-tert-butylphosphide ligands; in addition, each platinum center bears a terminal carbonyl ligand. This triplatinum cluster was initially developed in view of applications in the field of cluster-containing innovative materials. Yet, due to the large success of platinum complexes in cancer treatment, we also decided to explore its cytotoxic and anticancer properties. Accordingly, the solubility profile of this compound in several solvents was preliminarily investigated, revealing a conspicuous solubility in DMSO and DMSO/buffer mixtures; this makes the biological testing of I amenable. UV–Vis measurements showed that the triplatinum cluster is stable for several hours under a variety of conditions, within aqueous environments. No measurable reactivity was observed for I toward two typical model proteins, i.e. lysozyme and cytochrome c. On the contrary, a significant reactivity was evidenced when reacting I with small sulfur-containing ligands. In particular, a pronounced reactivity with reduced glutathione and cysteine emerged from ESI-MS experiments, proving complete formation of I-GSH and I-Cys derivatives, with the loss of a single carbonyl ligand. Starting from these encouraging results, the cytotoxic potential of I was assayed in vitro against a panel of representative cancer cell lines, and potent cytotoxic properties were disclosed. Of particular interest is the finding that the triplatinum species manifests potent antiproliferative properties toward Triple Negative Breast Cancer Cells, often refractory to most anticancer drugs. Owing to the reported encouraging results, a more extensive biological and pharmacological evaluation of this Pt cluster is now warranted to better elucidate its mode of action.

The bis carbene gold(I) complex [Au(1-butyl-3-methyl-2-ylidene)2]PF6, ([Au(NHC)2]PF6 hereafter), holds remarkable interest as a perspective anticancer agent. The compound is stable under physiological like conditions: its original structure is retained even in the presence of excess glutathione (GSH). Previous studies revealed its high cytotoxicity in vitro that correlates with the impairment of crucial metabolic and enzymatic cellular processes (Magherini et al., Oncotarget, 2018, 9, 28042). Here, the interaction of [Au(NHC)2]PF6 with the human telomeric DNA G-quadruplex Tel23 has been investigated in solution by means of high resolution mass spectrometry. ESI MS experiments well document the formation of stable 1 : 1 adducts between the biscarbene gold complex – in its intact form – and the DNA G-quadruplex Tel23. Next, through independent biophysical methods, we show that [Au(NHC)2]PF6 binding does not significantly affect the G quadruplex melting temperature nor its conformation. The crystal structure for the [Au(NHC)2]+/Tel24 adduct was eventually determined by a joint X-ray diffraction and in silico simulation approach. Through the careful integration of solution and solid-state data, a quite clear picture emerges for the interaction of this gold complex with the Tel23 G-quadruplex.

The antiproliferative properties of a group of 13 structurally diverse gold(III) compounds, including six mononuclear gold(III) complexes, five dinuclear oxo-bridged gold(III) complexes, and two organogold(III) compounds, toward several human tumor cell lines were evaluated in vitro using a systematic screening strategy. Initially all compounds were tested against a panel of 12 human tumor cell lines, and the best performers were tested against a larger 36-cell-line panel. Very pronounced antiproliferative properties were highlighted in most cases, with cytotoxic potencies commonly falling in the low micromolar—and even nanomolar—range. Overall, good-to-excellent tumor selectivity was established for at least seven compounds, making them particularly attractive for further pharmacological evaluation. Compare analysis suggested that the observed antiproliferative effects are caused by a variety of molecular mechanisms, in most cases "DNA-independent,” and completely different from those of platinum drugs. Remarkably, some new biomolecular systems such as histone deacetylase, protein kinase C/staurosporine, mammalian target of rapamycin/rapamycin, and cyclin-dependent kinases were proposed for the first time as likely biochemical targets for the gold(III) species investigated. The results conclusively qualify gold(III) compounds as a promising class of cytotoxic agents, of outstanding interest for cancer treatment, while providing initial insight into their modes of action. Graphical Abstract: A series of gold(III) compounds showed cytotoxic properties and tumor selectivity toward a panel of cancer cell lines. Compare analysis provided insight into their possible mechanisms of action

Correction for ‘Reactions of metallodrugs with proteins: selective binding of phosphane-based platinum(ii) dichlorides to horse heart cytochrome c probed by ESI MS coupled to enzymatic cleavage’ by Carolin Mügge et al., Metallomics, 2011, 3, 987–990.

The triplatinum complex of the 2,4,6-Tris(2-pyrimidyl)-1,3,5-triazine ligand, Pt 3 TPymT hereafter, has been prepared and characterized for the first time. NMR studies point out that the three platinum(II) centers possess an identical coordination environment. The interactions of Pt 3 TPymT with DNA were explored in comparison to the free ligand. Specifically, fluorescence, mass spectrometry, viscometry and melting measurements were carried out. In contrast to expectations, the obtained data reveal that no intercalative binding takes place; we propose that binding of Pt 3 TPymT to DNA mainly occurs through external/groove binding.

// Francesca Magherini 1 , Tania Fiaschi 1 , Elisa Valocchia 1 , Matteo Becatti 1 , Alessandro Pratesi 2 , Tiziano Marzo 2, 3 , Lara Massai 2 , Chiara Gabbiani 3 , Ida Landini 4 , Stefania Nobili 5 , Enrico Mini 5 , Luigi Messori 2 , Alessandra Modesti 1 and Tania Gamberi 1 1 Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, University of Florence, Florence, Italy 2 Department of Chemistry “Ugo Schiff”, University of Florence, Florence, Italy 3 Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa, Italy 4 Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy 5 Department of Health Sciences, University of Florence, Florence, Italy Correspondence to: Tania Gamberi, email: tania.gamberi@unifi.it Luigi Messori, email: luigi.messori@unifi.it Keywords: gold(I)-N-heterocyclic carbene complexes; A2780 human ovarian cancer cells; proteomics; mitochondrial metabolism; thioredoxin reductase Received: August 13, 2017 Accepted: May 19, 2018 Published: June 15, 2018 ABSTRACT Au(NHC) and Au(NHC) 2 , i.e. a monocarbene gold(I) complex and the corresponding bis(carbene) complex, are two structurally related compounds, endowed with cytotoxic properties against several cancer cell lines. Herein, we explore the molecular and cellular mechanisms at the basis of their cytotoxicity in A2780 human ovarian cancer cells. Through a comparative proteomic analysis, we demonstrated that the number of modulated proteins is far larger in Au(NHC) 2 -treated than in Au(NHC)-treated A2780 cells. Both gold compounds mainly affected proteins belonging to the following functional classes: protein synthesis, metabolism, cytoskeleton and stress response and chaperones. Particularly, Au(NHC) 2 gave rise to an evident upregulation of several glycolytic enzymes. Moreover, only Au(NHC) 2 triggered a net impairment of respiration and a metabolic shift towards glycolysis, suggesting that mitochondria are relevant cellular targets. We also found that both carbenes, similarly to the gold(I) compound auranofin, caused a strong inhibition of the seleno-enzyme thioredoxin reductase (TrxR). In conclusion, we highlighted that coordination of two carbene ligands to the same gold(I) center greatly enhances the antiproliferative effects of the resulting compound in comparison to the monocarbene derivative. Moreover, TrxR inhibition and metabolic impairment seem to play a major role in the Au(NHC) 2 cytotoxicity. Overall, these antiproliferative effects were also confirmed on other two human ovarian cancer cell lines ( i.e. SKOV3 and IGROV1).

A series of organometallic AuI N-heterocyclic carbene (NHC) complexes was synthesized and characterized for anticancer activity in four human cancer cell lines. The compounds' toxicity in healthy tissue was determined using precision-cut kidney slices (PCKS) as a tool to determine the potential selectivity of the gold complexes ex vivo. All evaluated compounds presented cytotoxic activity toward the cancer cells in the nano-or low micromolar range. The mixed AuI NHC complex, (tert-butylethynyl)-1,3-bis-(2,6-diisopropylphenyl) imidazol-2-ylidene gold(I), bearing an alkynyl moiety as ancillary ligand, showed high cytotoxicity in cancer cells in vitro, while being barely toxic in healthy rat kidney tissues. The obtained results open new perspectives toward the design of mixed NHC-alkynyl gold complexes for cancer therapy.

Aubipyc is an organogold(III) compound endowed with encouraging anti-proliferative properties in vitro that is being evaluated pre-clinically as a prospective anticancer agent. A classical proteomic approach is exploited here to elucidate the mechanisms of its biological actions in A2780 human ovarian cancer cells. Based on 2-D gel electrophoresis separation and subsequent mass spectrometry identification, a considerable number of differentially expressed proteins were highlighted in A2780 cancer cells treated with Aubipyc. Bioinformatic analysis of the groups of up-regulated and down-regulated proteins pointed out that Aubipyc primarily perturbs mitochondrial processes and the glycolytic pathway. Notably, some major alterations in the glycolytic pathway were validated through Western blot and metabolic investigations. Biological significance This is the first proteomic analysis regarding Aubipyc cytotoxicity in A2780/S ovarian cancer cell line. Aubipyc is a promising gold(III) compound which manifests an appreciable cytotoxicity toward the cell line A2780, being able to overcome resistance to platinum. The proteomic study revealed for Aubipyc different cellular alterations with respect to cisplatin as well as to other gold compound such as auranofin. Remarkably, the bioinformatic analysis of proteomic data pointed out that Aubipyc treatment affected, directly or indirectly, several glycolytic enzymes. These data suggest a new mechanism of action for this gold drug and might have an impact on the use of gold-based drug in cancer treatment.

A group of mixed-ligand Pt(II) complexes bearing acetylacetonate and sulphur ligands were recently developed in the University of Lecce as a new class of prospective anticancer agents that manifested promising pharma-cological properties in preliminary in vitro and in vivo tests. Though modelled on the basis of cisplatin, these Pt(II) complexes turned out to exhibit a profoundly distinct mode of action as they were found to act mainly on non-genomic targets rather than on DNA. Accordingly, we have explored here their reactions with two representative model proteins through an established ESI–MS procedure with the aim to describe their general interaction mechanism with protein targets. A pronounced reactivity with the tested proteins was indeed documented; the nature of the resulting metallodrug-protein interactions could be characterised in depth in the various cases. Preferential binding to protein targets compared to DNA is supported by independent ICP-OES measurements. The implications of these findings are discussed.

AuL10 [AuIIICl2DMDT] (DMDT: N,N-dimethyldithiocarbamate) and AuL12 [AuIIIBr2ESDT] (ESDT: ethylsarcosine dithiocarbamate) are two extremely promising gold-based anticancer compounds. Their mechanisms of action are still largely unknown although some specific hypotheses were put forward. To shed light on reactivity with probable biological targets we report here on their interactions with serum albuminand with calf thymus DNA taken as model biomolecules. Quite unexpectedly, spectrophotometric investigations revealed substantially different patterns of interaction for these two gold complexes with the mentioned biomolecules, probably arising from differences in redox chemistry. Afterwards, AuL12 was tested against A549 human non-small-cell lung carcinoma cells, evaluating its real-time profile of cell-growth inhibition by the xCELLigence Real-Time Cell Analysis (RTCA) system. Measures of the impedance at the base of each well by non-invasive gold microelectrodes point out that this gold complex inhibits cell proliferation after only 4 hours of treatment, producing its cytotoxic effects probably at the membrane level.

The bottom-up mass spectrometry approach is today one of the best tools of Metallomics to characterize the binding of metal-based drugs to proteins. Yet, the stability of metal-protein coordination bonds along the whole process may be a critical issue. This led us to build up a general protocol to test metallodrug-protein adduct stability under the typical conditions of the filter-aided sample preparation (FASP)/bottom-up procedure, ranging from the analysis of solutions containing metal-protein adducts to tandem mass spectrometry experiments. More in detail, we identified nine critical situations, either during the sample manipulations or instrumental, as a potential source of metal-protein bond impairment when using FASP operative conditions and a nano high performance liquid chromatography-nanoelectrospray ionization-LTQ-Orbitrap (nanoLC-nanoESI-LTQ-Orbitrap) mass spectrometer system, equipped with a preconcentration/purification device. These are: 1) sample permanence in the ammonium bicarbonate buffer; 2) denaturation with urea; 3) reduction with dithiothreitol; 4) alkylation with iodoacetamide; 5) sample permanence in the loading mobile phase; 6) sample permanence in the elution mobile phase; 7) the nanoESI process; 8) the transfer of the adduct through ion transfer tube and tube lens; 9) collision induced dissociation in the ion trap. Accordingly, an ad hoc experimental protocol was developed and applied to the adducts formed between cytochrome c (Cyt c) and two different metallodrugs, i.e. cisplatin (cis-diamminedichloridoplatinum(II), CDDP) and RAPTA-C, a well-known ruthenium(II)-arene compound [Ru(η6-p-cymene)Cl2(pta)] (pta=1,3,5-triaza-7-phosphaadamantane), used here as models. Notably, Cyt c-CDDP adducts were stable through all the above conditions while Cyt c-RAPTA-C adducts turned out unstable in the ammonium bicarbonate buffer. This latter finding supports the need to perform a test-protocol of this kind when starting any extensive bottom-up MS investigation of protein-metallodrug systems.

The X-ray structure of the adduct formed in the reaction between the gold N-heterocyclic carbene compound Au(NHC)Cl (with NHC = 1-butyl-3-methyl-imidazole-2-ylidene) and the model protein thaumatin is reported here. The structure reveals binding of Au(NHC)(+) fragments to distinct protein sites. Notably, binding of the gold compound occurs at lysine side chains and at the N-terminal tail; the metal binds the protein after releasing Cl(-) ligand, but retaining NHC fragment.

The dibromido analogue of cisplatin, cis-PtBr2(NH3)2 (cisPtBr2 hereafter), has been prepared and characterised. Its solution behaviour in standard phosphate buffer, at pH 7.4, was investigated spectrophotometrically and found to reproduce quite closely that of cisplatin; indeed, progressive sequential release of the two halide ligands typically occurs as in the case of cisplatin, with a roughly similar kinetics. Afterward, patterns of reactivity toward model proteins and standard ctDNA were explored and the nature of the resulting interactions elucidated. The antiproliferative properties were then evaluated in four representative cancer cell lines, namely A549 (human lung cancer), HCT116 (human colon cancer), IGROV-1 (human ovarian cancer) and FLG 29.1 (human acute myeloid leukaemia). Cytotoxic properties in line with those of cisplatin were highlighted. From these studies an overall chemical and biological profile emerges for cisPtBr2 closely matching that of cisplatin; the few slight, but meaningful differences that were underscored might be advantageously exploited for clinical application.

Alzheimer's disease (AD) is a widespread neurodegenerative disease with a very high medical, social and economic burden. The etiopathogenesis of AD is still largely obscure; however, there is growing evidence that aggregation of β-amyloid peptides (Aβ) into a variety of supramolecular structures is critically involved in its insurgence and progression (the so called “amyloid cascade hypothesis”). Recent results point to oligomeric Aβ aggregates rather than mature Aβ fibrils as the major culprit for neurotoxicity; details of the inherent aggregation processes are being progressively clarified. In view of these achievements, early stages of Aβ aggregation are considered today a realistic “druggable” target for the development of new anti-AD agents. Notably, a variety of organic compounds that are able to inhibit effectively Aβ aggregation represent promising drug candidates. Metal based compounds capable of interacting with the N-terminal metal binding site of amyloid peptides might similarly contrast metal-induced Aβ aggregation and serve as potential drugs for AD. In a recent pioneering study Barnham et al. showed that platinum(II) phenanthroline complexes strongly inhibit Aβ oligomerisation and attenuate its neurotoxicity in vitro. A number of additional examples involving metal complexes as inhibitors of Aβ aggregation were reported afterward. On the ground of those results it may be proposed that metal based compounds constitute today a suitable and rich source for novel anti-AD agents. The potential and the limits of this therapeutic option are comprehensively and critically discussed as well as the perspectives for future research.

Gold compounds are a class of metallodrugs with great potential for cancer treatment. During the last two decades, a large variety of gold(I) and gold(III) compounds are reported to possess relevant antiproliferative properties in vitro against selected human tumor cell lines, qualifying themselves as excellent candidates for further pharmacological evaluation. The unique chemical properties of the gold center confer very interesting and innovative pharmacological profiles to gold-based metallodrugs. The primary goal of this review is to define the state of the art of preclinical studies on anticancer gold compounds, carried out either in vitro or in vivo. The available investigations of anticancer gold compounds are analyzed in detail, and particular attention is devoted to underlying molecular mechanisms. Notably, a few biophysical studies reveal that the interactions of cytotoxic gold compounds with DNA are generally far weaker than those of platinum drugs, implying the occurrence of a substantially different mode of action. A variety of alternative mechanisms were thus proposed, of which those involving either direct mitochondrial damage or proteasome inhibition or modulation of specific kinases are now highly credited. The overall perspectives on the development of gold compounds as effective anticancer drugs with an innovative mechanism of action are critically discussed on the basis of the available experimental evidence.

The reaction of bovine serum albumin (BSA) with [ trans-RuCl 4(Im)(dimethylsulfoxide)][ImH] (Im = imidazole) (NAMI-A), an experimental ruthenium(III) anticancer drug, and the formation of the respective NAMI-A/BSA adduct were investigated by X-ray absorption spectroscopy (XAS) at the sulfur and chlorine K-edges and at the ruthenium K- and L 3-edges. Ruthenium K and L 3-edge spectra proved unambiguously that the ruthenium center remains in the oxidation state +3 after protein binding. Comparative analysis of the chlorine K-edge XAS spectra of NAMI-A and NAMI-A/BSA, revealed that the chlorine environment is greatly perturbed upon protein binding. Only modest changes were observed in the sulfur K-edge spectra that are dominated by several protein sulfur groups. Overall, valuable information on the nature of this metallodrug/protein adduct and on the mechanism of its formation was gained; XAS spectroscopy turns out to be a very suitable method for the characterization of this kind of systems.

The electrostatic surface of cytochrome c and its changes with the iron oxidation state are involved in the docking and undocking processes of this protein to its biological partners in the mitochondrial respiratory pathway. To investigate the subtle mechanisms of formation of productive macromolecular complexes and of their breakage following the electron transfer process, the X-ray structures of horse heart ferri-cytochrome c (trigonal form) and ferro-cytochrome c (monoclinic form) were obtained using nitrate ions both as a crystallizing agent and an anionic probe for mapping the electrostatic surface changes. Both crystal forms contain three protein molecules in the asymmetric unit. In addition, a total of 21.5 and 18 crystallographically independent nitrate ions were identified for the trigonal and monoclinic forms, respectively. By matching all the six crystallographically independent protein molecules, 26 different anion-protein interaction sites were identified on the surfaces of cytochrome c, 10 of which were found in both forms, 8 present only in the oxidized and 8 only in the reduced form. The structural analysis of the electron transfer complexes, based on this new information, suggests a specific exit strategy for cytochrome c after formation of productive protein-protein complexes: a directional sliding mechanism for the electron shuttle on the surface of the redox partner is proposed to take place after the electron transfer process has occurred.

A novel "Keppler type" ruthenium(III) compound trans-[bis(2-amino 5-methylthiazole)tetrachlororuthenate(III)] 1, of potential interest as an anticancer agent, was designed, synthesized, and characterized. Its interactions with various proteins were analyzed, including the selenoenzyme thioredoxin reductase, an emerging target for anticancer metallodrugs. The selective inhibition of the cytosolic form of this selenoenzyme was documented, this being the first report of significant thioredoxin reductase inhibition by a ruthenium compound.

The investigation of cis-PtI2(NH3)2, the diiodido analogue of cisplatin (cisPtI2 hereafter), has been unjustly overlooked so far mainly because of old claims of pharmacological inactivity. Some recent - but still fragmentary - findings prompted us to reconsider more systematically the chemical and biological profile of cisPtI2 in comparison with cisplatin. Its solution behaviour, interactions with DNA and cytotoxic properties versus selected cancer cell lines were thus extensively analysed through a variety of biophysical and computational methods. Notably, we found that cisPtI2 is highly cytotoxic in vitro toward a few solid tumour cell lines and that its DNA platination pattern closely reproduces that of cisplatin; cisPtI2 is also shown to completely overcome resistance to cisplatin in a platinum resistant cancer cell line. The differences in the biological actions of these two Pt complexes are most likely related to slight but meaningful differences in their solution behaviour and reactivity. Overall, a very encouraging and unexpected pharmacological profile emerges for cisPtI2 with relevant implications both in terms of mechanistic knowledge and of prospective clinical application. An ab initio DFT study is also included to support the interpretation of the solution behaviour of cisPtI2 under physiological and slightly acidic pH conditions, TM and LM acknowledge Beneficentia Stiftung (Vaduz), COST Action CM1105 and AIRC (IG-16049) for generous financial support. JK and VB acknowledge the support from the Czech Science Foundation (Grant 14-21053S)

The tryptic digests of blood samples obtained from transferrin C1 and C2 (TfC1 and TfC2 hereafter) genotypes were analysed by Liquid Chromatography coupled to Electrospray Mass Spectrometry (LC/ESI–MS/MS). The analytical results confirmed the single base change in exon 15 of the Tf gene. The solution behaviour and the iron binding properties of the two Tf variants were studied by UV-visible spectrophotometry and by circular dichroism. It appears that TfC2 globally manifests the same spectral features as the native protein. The local conformation of the two iron binding sites is conserved in the two Tf variants as evidenced by the visible absorption and CD spectra. Also, the iron binding capacities and their pH-dependent profiles are essentially the same. Overall, our investigation points out that the single amino acid substitution in TfC2 (Pro570Ser) does not affect the general conformation of the protein nor the local structure of the iron binding sites. The implications of these results for the etiopathogenesis of Alzheimer's disease are discussed.

The cellular alterations produced in cisplatin-resistant A2780 ovarian cancer cells (A2780/R) upon treatment with the cytotoxic organogold(III) complex Aubipyc were investigated in depth through a classical proteomic approach. We observed that A2780/R cell exposure to a cytotoxic concentration of Aubipyc for 24 hours results in a conspicuous number of alterations at the protein level that were carefully examined. Notably, we observed that several affected proteins belong to the glucose metabolism system further supporting the idea that the cytotoxic effects of Aubipyc in A2780/R cells are mostly mediated by an impairment of glucose metabolism in excellent agreement with previous observations on the parent cisplatin-sensitive cell line.

A 13-membered ring cyclic tetrapeptide was synthesized by the solid-phase peptide synthesis method, and its copper(II) coordination properties were analyzed by optical spectroscopy, mass spectrometry, and electrochemistry. All collected data strongly support the presence, at alkaline pH, of a stable peptide/copper(III) complex that is formed in solution by atmospheric dioxygen oxidation. On the basis of previous studies on cyclic peptide/copper systems, we suggest that the copper(III) ion is at the center of the ligand's cavity being coordinated to four deprotonated amide nitrogen atoms. This donor set would greatly lower the redox potential for the CuIII/CuII couple, thus allowing easy oxidation of the coordinated copper(II) by atmospheric oxygen.

The Keppler-type compound, PMRU20, utilized in this work, was first prepared by Dr. Pasquale Mura, Institute of Crystallography, CNR, Montelibretti Rome, a few years ago. The synthesis and characterization of this compound, both in solid state and in solution, are reported in the paper quoted here as ref 17 (Mura, P.; Piccioli, F.; Gabbiani, C.; Camalli, M.; Messori, L. Inorg. Chem.2005, 44, 4897–4899) resulting from a collaboration between Institute of Crystallography, CNR, Montelibretti Rome, and the Department of Chemistry, University of Florence. We gratefully acknowledge Dr. Pasquale Mura for the gift of a PMRU20 sample.

Metal complexes represent today an attractive class of experimental anti-Alzheimer agents with the potential of blocking β-amyloid 1-42 aggregation and scavenging its toxicity. Three representative ruthenium(III) complexes, namely NAMI A, KP1019, and PMRU20, were specifically evaluated to this end in an established in vitro model of AD relying on primary cortical neurons. Notably, PMRU20 turned out to be highly effective in protecting cortical neurons against Aβ 1-42 toxicity, while the other tested ruthenium compounds were poorly active or even inactive; we also found that PMRU20 is virtually devoid of any significant toxicity in vitro at the applied concentrations. Interestingly, PMRU20 was neuroprotective even against the toxicity induced by Aβ 25-35. The direct reaction of PMRU20 with Aβ 1-42 was explored through ESI MS analysis and some adduct formation evidenced. In addition, thioflavin T assays revealed that PMRU20 greatly reduces Aβ 1-42 aggregation. The implications of these findings are discussed in relation to emerging treatment strategies for the Alzheimer's disease.

International audience; : Picking a fight with parasites! Trypanothione reductase (TR) is a validated drug target for the development of antileishmanial agents. A group of structurally diverse gold-containing compounds was evaluated in vitro for TR inhibition. A number of compounds exhibited potent activity and deserve further pharmacological evaluation.

Research has increasingly focused on cytotoxic gold and ruthenium compounds as anticancer drug candidates. From proteomic investigations, clearly emerged that a few different cellular pathways relevant for the comprehension of the pharmacological actions are specifically modulated by them. To gain a better intepretation of their cellular effects, we decided to undertake a comprehensive bioinformatic analysis of the available proteomic results. Data obtained from prevously published treatments were grouped and mapped in the PPI Spider on the web portal Bioprofiling (http://www.BioProfiling.de/gene_list). A preliminary map of protein-protein interactions was built up, and some mechanistically relevant features highlighted. In total, 34 proteins resulted to be direct gold and ruthenium compounds interactors; we built a statistically significant interaction network that grouped together all the proteins differentially expressed. Moreover, we showed as intermediate protein cREL a Component of the NF-kappa-B. This study explores the affected protein pathways from an interactomic prospective stressing the importance of advanced bioinformatic analysis.

Cytotoxic gold compounds hold today great promise as new pharmacological agents for treatment of human ovarian carcinoma; yet, their mode of action is still largely unknown. To shed light on the underlying molecular mechanisms, we performed 2D-DIGE analysis to identify differential protein expression in a cisplatin-sensitive human ovarian cancer cell line (A2780/S) following treatment with two representative gold(iii) complexes that are known to be potent antiproliferative agents, namely AuL12 and Au(2)Phen. Software analysis using DeCyder was performed and few differentially expressed protein spots were visualized between the three examined settings after 24 h exposure to the cytotoxic compounds, implying that cellular damage at least during the early phases of exposure is quite limited and selective, reflecting the attempts of the cell to repair damage and to survive the insult. The potential of novel proteomic methods to disclose mechanistic details of cytotoxic metallodrugs is herein further highlighted. Different patterns of proteomic changes were highlighted for the two metallodrugs with only a few perturbed protein spots in common. Using MALDI-TOF MS and ESI-Ion trap MS/MS, several differentially expressed proteins were identified. Two of these were validated by western blotting: Ubiquilin-1, responsible for inhibiting degradation of proteins such as p53 and NAP1L1, a candidate marker identified in primary tumors. Ubiquilin-1 resulted over-expressed following both treatments and NAP1L1 was down-expressed in AuL12-treated cells in comparison with control and with Au(2)Phen-treated cells. In conclusion, we performed a comprehensive analysis of proteins regulated by AuL12 and Au(2)Phen, providing a useful insight into their mechanisms of action.

Amyloid-β(1-42) (Aβ) is believed to play a crucial role in the ethiopathogenesis of Alzheimer's Disease (AD). In particular, its interactions with biologically relevant metal ions may lead to the formation of highly neurotoxic complexes. Here we describe the species that are formed upon reacting Aβ with several biometals, namely copper, zinc, iron, and with non-physiological aluminum to assess whether different metal ions are able to differently drive Aβ aggregation. The nature of the resulting Aβ-metal complexes and of the respective aggregates was ascertained through a number of biophysical techniques, including electrospray ionization mass spectrometry, dynamic light scattering, fluorescence, transmission electron microscopy and by the use of conformation-sensitive antibodies (OC, αAPF). Metal binding to Aβ is shown to confer highly different chemical properties to the resulting complexes; accordingly, their overall aggregation behaviour was deeply modified. Both aluminum(III) and iron(III) ions were found to induce peculiar aggregation properties, ultimately leading to the formation of annular protofibrils and of fibrillar oligomers. Notably, only Aβ-aluminum was characterized by the presence of a relevant percentage of aggregates with a mean radius slightly smaller than 30 nm. In contrast, both zinc(II) and copper(II) ions completely prevented the formation of soluble fibrillary aggregates. The biological effects of the various Aβ-metal complexes were studied in neuroblastoma cell cultures: Aβ-aluminum turned out to be the only species capable of triggering amyloid precursor and tau181 protein overproduction. Our results point out that Al can effectively interact with Aβ, forming "structured" aggregates with peculiar biophysical properties which are associated with a high neurotoxicity.

The reactions of human beta-amyloid peptide 1-28 (A beta 28) with Al-III and Fe-III ions were investigated by H-1 NMR and electrospray ionization mass spectrometry (ESI-MS) under pH conditions close to physiological ones. H-1 NMR titrations, performed in the 5.3-8.0 pH range, revealed that no measurable amounts of A beta 28-Al-III or A beta 28-Fe-III adducts are formed; such metal adducts could not be obtained even by changing a number of experimental conditions, e.g., temperature, buffer, nature of the salt, etc. These observations were later confirmed by ESI-MS. It is thus demonstrated that A beta 28, at physiological pH, is not able to form binary complexes with Al-III and Fe-III ions of sufficient stability to compete with metal hydroxide precipitation. The biological implications of these findings are discussed in the frame of current literature.

A novel dioxo-bridged dinuclear gold(III) complex with two 2,9-dimethylphenanthroline ligands was synthesized and thoroughly characterized. Its crystal structure was solved, and its solution behavior assessed. Remarkably, this compound revealed excellent antiproliferative properties in vitro against a wide panel of 36 cancer cell lines, combining a high cytotoxic potency to pronounced tumor selectivity. Very likely, these properties arise from an innovative mode of action (possibly involving histone deacetylase inhibition), as suggested by COMPARE analysis. In turn, electrospray ionization−mass spectrometry studies provided valuable insight into its molecular mechanisms of activation and of interaction with protein targets. Gold(III) reduction, dioxo bridge disruption, coordinative gold(I) binding to the protein, and concomitant release of the phenanthroline ligand were proposed to occur upon interaction with superoxide dismutase, used here as a model protein. Because of the reported results, this new gold(III) compound qualifies itself as an optimal candidate for further pharmacological testing.

Since the discovery of cisplatin and its introduction in the clinics, metal compounds have been intensely investigated in view of their possible application in cancer therapy. In this frame, a deeper understanding of their mode of action, still rather obscure, might turn crucial for the design and the obtainment of new and better anticancer agents. Due to the extreme complexity of the biological systems, it is now widely accepted that innovative and information-rich methods are absolutely needed to afford such a goal. Recently, both proteomic and metallomic strategies were successfully implemented for the elucidation of specific mechanistic features of anticancer metallodrugs within an innovative “Systems Biology” perspective. Particular attention was paid to the following issues: i) proteomic studies of the molecular basis of platinum resistance; ii) proteomic analysis of cellular responses to cytotoxic metallodrugs; iii) metallomic studies of the transformation and fate of metallodrugs in cellular systems. Notably, those pioneering studies, that are reviewed here, allowed a significant progress in the understanding of the molecular mechanisms of metal based drugs at the cellular level. A further extension of those studies and a closer integration of proteomic and metallomic strategies and technologies might realistically lead to rapid and significant advancements in the mechanistic knowledge of anticancer metallodrugs.