Chiara Gabbiani

The thioredoxin system plays a key role in regulating the overall intracellular redox balance. It basically comprises the small redox protein thioredoxin (Trx), nicotinamide adenine dinucleotide phosphate, in its reduced form (NADPH), and thioredoxin reductase (TrxR), a large homodimeric selenzoenzyme controlling the redox state of thioredoxin. Details of the thioredoxin system are provided herein, particular emphasis being given to the protein chemistry of thioredoxin reductases. Several lines of evidence point out today that the thioredoxin system represents an effective "druggable" target for the development of new anticancer agents. Accordingly, a number of established anticancer agents were retrospectively found to be potent inhibitors of thioredoxin reductases and to induce severe oxidative stress. During the last decade a variety of gold compounds, either gold(I) or gold(III), were reported to manifest outstanding antitumor properties, forming a promising class of experimental anticancer agents. In turn, recent studies have revealed that several cytotoxic gold compounds, either gold(I) or gold(III), are potent TrxR inhibitors. Details of their mechanism of selenoenzyme inhibition are currently under investigation, in our laboratory, and some new results will be anticipated here; notably, preferential gold targeting of active site selenolate could be experimentally supported. Based on the numerous experimental evidences now available, both at the molecular and cellular level, we propose that the relevant cytotoxic actions produced by gold compounds are mainly the result of potent inhibition of thioredoxin reductase; the alterations of mitochondrial functions, elicited by profound TrxR inhibition, would eventually lead to cell apoptosis. A general and unitary framework is thus offered to interpret the mode of action of cytotoxic gold compounds, according to which they should be primarily considered as antimitochondrial drugs. The peculiar properties of gold compounds highlighted in this review might be further exploited for the obtainment of newer and selective anticancer agents.

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.

A series of ruthenium(II)-arene (RAPTA) compounds were evaluated for their ability to inhibit thioredoxin reductase (either cytosolic or mitochondrial) and cathepsin B, two possible targets for anticancer metallodrugs. In general, inhibition of the thioredoxin reductases was lower than that of cathepsin B, although selected compounds were excellent inhibitors of both classes of enzymes in comparison to other metal-based drugs. Some initial structure-activity relationships could be established. On the basis of the obtained data, different mechanisms of binding/inhibition appear to be operative; remarkably the selectivity of the ruthenium compounds toward solid metastatic tumors also correlates to the observed trends. Notably, docking studies of the interactions of representative RAPTA compounds with cathepsin B were performed that provided realistic structures for the resulting protein-metallodrug adducts. Good agreement was generally found between the inhibiting potency of the RAPTA compounds and the computed stability of the corresponding cat B/RAPTA adducts.

Gold(III) compounds constitute an emerging class of biologically active substances, of special interest as potential anticancer agents. During the past decade a number of structurally diverse gold(III) complexes were reported to be acceptably stable under physiological-like conditions and to manifest very promising cytotoxic effects against selected human tumour cell lines, making them good candidates as anti-tumour drugs. Some representative examples will be described in detail. There is considerable interest in understanding the precise biochemical mechanisms of these novel cytotoxic agents. Based on experimental evidence collected so far we hypothesize that these metallodrugs, at variance with classical platinum(II) drugs, produce in most cases their growth inhibition effects through a variety of “DNA-independent” mechanisms. Notably, strong inhibition of the selenoenzyme thioredoxin reductase and associated disregulation of mitochondrial functions were clearly documented in some selected cases, thus providing a solid biochemical basis for the pronounced proapoptotic effects. These observations led us to investigate in detail the reactions of gold(III) compounds with a few model proteins in order to gain molecular-level information on the possible interaction modes with possible protein targets. Valuable insight on the formation and the nature of gold–protein adducts was gained through ESI MS (electrospray ionization mass spectrometry) and spectrophotometric studies of appropriate model systems as it is exemplified here by the reactions of two representative gold(III) compounds with cytochrome c and ubiquitin. The mechanistic relevance of gold(III)-induced oxidative protein damage and of direct gold coordination to protein sidechains is specifically assessed. Perspectives for the future of this topics are briefly outlined.

Tropical diseases today constitute a major health problem and a big challenge for drug discovery. Because of the limited arsenal of effective antiparasitic agents and the frequent appearance of chemoresistance, there is an urgent and continuous need to develop new drugs against these ailments. Metal compounds still offer excellent opportunities to find new ‘leads’ against the major protozoan diseases such as malaria, leishmaniasis and trypanosomiasis. A few metal-based drugs are already available in this therapeutic area, and others are currently being developed. Recent progress in parasite genomics and the identification of a few biomolecular targets hold great promise for the discovery of new ‘mechanism-based’ antiparasitic metallodrugs. The trends and perspectives for this exciting research field are outlined here.

A series of six dinuclear gold(III) oxo complexes with bipyridyl ligands, of general formula [Au2(N,N)2(mu-O)2][PF6]2 (Auoxo1-Auoxo6) [where N,N = 2,2'-bipyridine (1), 4,4'-di-tert-butyl- (2), 6-methyl- (3), 6-neopentyl- (4), 6-(2,6-dimethylphenyl)- (5), 6,6'-dimethyl-2,2'-bipyridine (6)], were investigated as potential cytotoxic and anticancer agents, and their antiproliferative properties were evaluated in vitro toward the reference A2780 human ovarian carcinoma cell line. While five compounds manifested moderate cytotoxic properties (with IC50 approximately 10-30 microM), the sixth one (Auoxo6), turned out to be approximately 5-15 times more active against both cell lines and will merit further pharmacological studies. The interactions of Auoxo1 and Auoxo6 with a few model proteins (serum albumin, cytochrome c, ubiquitin) and with calf thymus DNA were analyzed in detail by various spectroscopic methods. Both tested compounds manifested a high and peculiar reactivity toward the mentioned model proteins; specific differences were detected in their reactivity with DNA. The mechanistic implications of these results are discussed.

We report an investigation on the effects of counterions of magnesium salts on the homogeneous phase precipitation reaction to yield nanoparticles. The results provide new parameters to guide the control of the size, monodispersity, crystallinity, and morphology of Mg(OH)2 nanoparticles. Evidence emerged that magnesium counterions affect the size of crystallites. The particle size increases from about 50 to 200 nm and follows the Hofmeister anion series: sulfate < chloride < nitrate ≤ perchlorate. All of the results show that a proper choice of counterion and composition of reaction mixture allows one to modulate and set conditions that predetermine production of nanoparticles with desired size. The Mg(OH)2 nanoparticles have been applied to the successful conservation of paper. To demonstrate the efficacy of nanoparticles as deacidifying agent and as protection against cellulose aging, some samples of paper from the 18th century were treated with the nanoparticles and artificially aged by subjecting them to hydrothermal and photooxidative degradation. The treatment with nanoparticles preserved the mechanical features of paper, and the deterioration was consistently reduced. The comparison with the Wei t'O method (based on magnesium alkoxides), one of the most common and largely used methods for paper deacidification, indicates the following: (1) nanoparticles present a higher efficacy in the deacidification treatment since they are much more reactive; (2) nanoparticles are less aggressive since they are easily converted into the carbonate form; (3) they present minor disadvantages related to the chemical nature of the solvent used in the Wei t'O (i.e., CFC); (4) papers/books can be treated with very simple procedures, and do not require any special apparatus; (5) nanoparticle treatments have substantial economic benefits.

The clinically established gold‐based antiarthritic drug auranofin (AF) manifests a pronounced reactivity toward thiol and selenol groups of proteins. In particular, AF behaves as a potent inhibitor of mammalian thioredoxin reductases causing severe intracellular oxidative stress. Given the high sensitivity of Plasmodium falciparum to oxidative stress, we thought that auranofin might act as an effective antimalarial agent. Thus, we report here new experimental results showing that auranofin and a few related gold complexes strongly inhibit P. falciparum growth in vitro . The observed antiplasmodial effects probably arise from direct inhibition of P. falciparum thioredoxin reductase. The above findings and the safe toxicity profile of auranofin warrant rapid evaluation of AF for malaria treatment in animal models.

Amyloid-β1–42 (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 interactions of cisplatin and its analogues, transplatin, carboplatin and oxaliplatin, with hen egg white lysozyme were analysed through ESI mass spectrometry, and the resulting metallodrug–protein adducts identified; the X-ray crystal structure of the cisplatin lysozyme derivative, solved at 1.9 Å resolution, reveals selective platination of imidazole Nε of His15.

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.

The solution behavior of auranofin, Et 3 PAuCl and Et 3 PAuI, 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 Et 3 PAuI confirmed that, similarly to auranofin, this drug is well tolerated in a murine model.Overall, a very similar profile emerges for Et 3 PAuI and Et 3 PAuCl, 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 such as lipophilicity could be exploited to improve bioavailability, with no loss of the pharmacological effects.

Gold-based drugs typically behave as strong inhibitors of the enzyme thioredoxin reductase (hTrxR), possibly as the consequence of direct Gold(I) coordination to its active site selenocysteine. To gain a deeper insight into the molecular basis of enzyme inhibition and prove gold-selenocysteine coordination, the reactions of three parent Gold(I) NHC compounds with the synthetic C-terminal dodecapeptide of hTrxR containing Selenocysteine at position 498, were investigated by electrospray ionization mass spectrometry (ESI-MS). Formation of 1:1 Gold-peptide adducts, though in highly different amounts, was demonstrated in all cases. In these adducts the same [Au-NHC]+ moiety is always associated to the intact peptide. Afterward, tandem MS experiments, conducted on a specific Gold-peptide complex, pointed out that Gold is coordinated to the selenolate group. The relatively large strength of the Gold-selenolate coordinative bond well accounts for potent enzyme inhibition typically afforded by these Gold(I) compounds. In a selected case, the time course of enzyme inhibition was explored. Interestingly, enzyme inhibition turned out to show up very quickly and reached its maximum just few minutes after mixing. Overall, the present results offer some clear insight into the process of thioredoxin reductase inhibition by Gold-based compounds.

Gold(III) complexes constitute a new class of metallodrugs, of potential interest for cancer treatment. During the past decade different kinds of gold(III) complexes have been reported to be appreciably stable under physiological-like conditions and to manifest relevant antiproliferative properties against selected human tumor cell lines. Some relevant examples are presented. Recent investigations point out that the interactions of cytotoxic gold(III) complexes with DNA are significantly different and weaker than those of platinum analogues; important interactions with model proteins and target proteins have been reported as well. Accordingly, the mechanisms of action of cytotoxic gold(III) complexes seem to be innovative and substantially different from that of cisplatin. Relevant antimitochondrial effects were demonstrated in some cases, eventually leading to cell apoptosis.

The effects of a few cytotoxic organogold(III) compounds on ovarian A2780 human cancer cells were investigated in comparison to cisplatin and oxaliplatin. The tested compounds produced significant antiproliferative effects and promoted apoptosis to a greater extent than platinum drugs while causing only modest cell cycle modifications. The mechanistic implications of these findings are discussed: mitochondrial pathways are proposed to be directly involved in the apoptotic process in relation to selective inhibition of thioredoxin reductase.

Clioquinol (CQ) is a "metal protein attenuating compound" that crosses the blood-brain barrier and binds, with high affinity, copper(II) and zinc(II), two metal ions critically involved in amyloid-beta aggregation and toxicity. CQ was recently proposed for the treatment of Alzheimer's disease, but controversial data have been reported so far concerning its real therapeutic advantages. We describe here results of chronic CQ treatment in the TgCRND8 mouse model of Alzheimer's disease. Remarkably, based on classical behavioral tests, CQ treatment was found to reverse, to a large extent, the working memory impairments that are characteristic of this mouse model. Pairwise, a significant reduction of amyloid-beta plaque burden, both in the cortex and in the hippocampus, was detected as well as an attenuation of astrogliosis. MALDI Mass Spectrometry Imaging technique revealed a specific localization of CQ in the above mentioned brain areas. Modest but significant effects on the absolute and relative brain concentrations of the three most important biometals (i.e., copper, zinc, and iron) were highlighted following CQ treatment. The pharmacological and mechanistic implications of the above findings are thoroughly discussed.

A series of structurally related oxo-bridged binuclear gold(III) compounds, [Au2(mu-O)2(N;N)2](PF6)2, where N;N is 2,2'-bipyridine or a substituted 2,2'-bipyridine, have recently been shown to exhibit appreciable stability under physiological-like conditions and to manifest important antiproliferative effects toward selected human tumor cell lines (J. Med. Chem. 2006, 49, 5524). The crystal structures of four members of this series, namely, [Au2(mu-O)2(bipy)2](PF6)2, cis-[Au2(mu-O)2(6-Mebipy)2](PF6)2, trans-[Au2(mu-O)2(6-oXylbipy)2](PF6)2, and [Au2(mu-O)2(6,6'-Me2bipy)2](PF6)2, have been solved here and the respective structural parameters comparatively analyzed. Remarkably, all of the compounds contain a common structural motif consisting of a Au2O2 "diamond core" linked to two bipyridine ligands in a roughly planar arrangement. Interestingly, introduction of different kinds of alkyl or aryl substituents on the 6 (and 6') position(s) of the bipyridine ligand leads to small structural changes that nonetheless greatly affect the reactivity of the metal centers. The chemical behavior of these compounds in solution has been studied in detail, focusing in particular on the electrochemical properties. Some initial correlations among the structural parameters, the chemical behavior in solution, and the known cytotoxic effects of these compounds are proposed. Notably, we have found that the 6,6'-dimethyl-2,2'-bipyridine derivative, which showed the largest structural deviations with respect to the model compound [Au2(mu-O)2(bipy)2](PF6)2, also had the highest oxidizing power, the least thermal stability, and the greatest cytotoxic activity. The positive correlation that exists between the oxidizing power and the antiproliferative effects seems to be of particular interest. Moreover, the electronic structures of these compounds were extensively analyzed using DFT methods, and the effects of the various substituents on reactivity were predicted; overall, very good agreement between theoretical expectations and experimental data was achieved. In turn, theoretical predictions offer interesting hints for the design of new, more active binuclear gold(III) compounds.

Electrospray ionisation mass spectrometry was used to analyse the reactions of metal compounds with mixtures of selected proteins. Three representative medicinally relevant compounds, cisplatin, transplatin and the organometallic ruthenium compound RAPTA-C, were reacted with a pool of three proteins, ubiquitin, cytochrome c and superoxide dismutase, and the reaction products were analysed using high-resolution mass spectrometry. Highly informative electrospray ionisation mass spectra were acquired following careful optimisation of the experimental conditions. The formation of metal-protein adducts was clearly observed for the three proteins. In addition, valuable information was obtained on the nature of the protein-bound metallofragments, on their distribution among the three different proteins and on the binding kinetics. The platinum compounds were less reactive and considerably less selective in protein binding than RAPTA-C, which showed a high affinity towards ubiquitin and cytochrome c, but not superoxide dismutase. In addition, competition studies between cisplatin and RAPTA-C showed that the two metallodrugs have affinities for the same amino acid residues on protein binding.

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 236IGEHMEEHGIK246 – 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.

Formation of amyloid-beta (Abeta){1-42} amyloid fibrils, a characteristic feature of Alzheimer's disease (AD), was monitored in situ through atomic force microscopy (AFM). Well-structured amyloid fibrils slowly formed in solution within 24 hours for which high quality AFM pictures could be obtained. Remarkably, addition of either copper(II) or zinc(II) ions to the incubation medium, even at extremely low molar ratios, dramatically changed the Abeta {1-42} aggregation profile and prevented fibril formation. Aggregates of different morphology appeared in accordance with previous observations: small globular aggregates upon addition of zinc; ill-structured micro-aggregates in the case of copper. The implications of these AFM results are discussed in the context of current concepts for AD metallobiology.

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.

Two novel gold carbene compounds, namely, chlorido (1-butyl-3-methyl-imidazole-2-ylidene) gold(I) (1) and bis(1-butyl-3-methyl-imidazole-2-ylidene) gold(I) (2), were prepared and characterized as prospective anticancer drug candidates. These compounds consist of a gold(I) center linearly coordinated either to one N-heterocyclic carbene (NHC) and one chloride ligand (1) or to two identical NHC ligands (2). Crystal structures were solved for both compounds, the resulting structural data being in good agreement with expectations. We wondered whether the presence of two tight carbene ligands in 2 might lead to biological properties distinct from those of the monocarbene complex 1. Notably, in spite of their appreciable structural differences, these two compounds manifested similarly potent cytotoxic actions in vitro when challenged against A2780 human ovarian carcinoma cells. In addition, both were able to overcome resistance to cisplatin in the A2780R line. Solution studies revealed that these gold carbene complexes are highly stable in aqueous buffers at physiological pH. Their reactivity with proteins was explored: no adduct formation was detected even upon a long incubation with the model proteins cytochrome c and lysozyme; in contrast, both compounds were able to metalate, to a large extent, the copper chaperone Atox-1, bearing a characteristic CXXC motif. The precise nature of the resulting gold-Atox-1 adducts was elucidated through ESI-MS analysis. On the basis of these findings, it is proposed that the investigated gold(I) carbene compounds are promising antiproliferative agents warranting a wider pharmacological evaluation. Most likely these gold compounds produce their potent biological effects through selective metalation and impairment of a few crucial cellular proteins.

The interactions between a few representative gold-based drugs and hen egg white lysozyme were studied by X-ray crystallography. High resolution crystal structures solved for three metallodrug–protein adducts provide valuable insight into the molecular mechanism of these promising metal compounds and the inherent protein metalation processes.

A variety of gold(III) and gold(I) derivatives of 2-(2′-pyridyl)benzimidazole (pbiH) were synthesized and fully characterized and their antiproliferative properties evaluated in a representative ovarian cancer cell line. The complexes include the mononuclear species [(pbi)AuX2] (X = Cl, 1; OAc, 2), [(pbiH)AuCl] (3), [(pbiH)Au(PPh3)][PF6] (4-PF6), and [(pbi)Au(L)] (L = PPh3, 5; TPA, 6), and the binuclear gold(I)/gold(I) and gold(I)/gold(III) derivatives [(PPh3)2Au2(μ2-pbi)][PF6] (10-PF6), [ClAu(μ3-pbi)AuCl2] (7),and [(PPh3)Au(μ3-pbi)AuX2][PF6] (X = Cl, 8-PF6; OAc, 9-PF6). The molecular structures of 6, 7, and 10-PF6 were determined by X-ray diffraction analysis. The chemical behavior of these compounds in solution was analyzed both by cyclic voltammetry in DMF and absorption UV–vis spectroscopy in an aqueous buffer. Overall, the stability of these gold compounds was found to be acceptable for the cellular studies. For all complexes, relevant antiproliferative activities in vitro were documented against A2780 human ovarian carcinoma cells, either resistant or sensitive to cisplatin, with IC50 values falling in the low micromolar or even in the nanomolar range. The investigated gold compounds were found to overcome resistance to cisplatin to a large degree. Results are interpreted and discussed in the frame of current knowledge on cytotoxic and antitumor gold compounds.

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.

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).

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.

Under the right conditions, as shown by high-quality ESI-MS spectra, a variety of platinum–protein adducts are formed from the reaction of various platinum drugs with cytochrome c. Met65 is proposed as the primary site for platinum coordination. Direct comparative information was obtained on the reactivity of these classical anticancer platinum compounds with proteins, and the mechanistic and methodological implications of these findings are discussed. Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Electrospray ionization mass spectrometry allows a rapid characterisation of the adducts formed between three novel anticancer ruthenium(II)-arene PTA compounds and horse heart cytochrome c or hen egg white lysozyme. Specific information on the nature of the protein-bound metallic fragments and the extent of protein metallation was readily obtained. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2452/2007/z600258_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

There is considerable interest today for the reactions of anticancer metallodrugs with proteins as these interactions might feature processes that are crucial for the biodistribution, the toxicity and even the mechanism of action of this important group of anticancer agents. We survey here the results of research activities carried out in our "Laboratory of Metals in Medicine" (Department of Chemistry, University of Florence) during the last three years, concerning the molecular characterisation of adducts formed between platinum, ruthenium and gold metallodrugs and a few model proteins. Valuable structural and functional information on these adducts could be derived from several biophysical studies mainly relying on the application of X-ray diffraction and ESI MS techniques. The value and the limitations of both approaches are outlined through a number of examples. Remarkably, the structural and functional information achieved on the respective metallodrug-protein adducts allowed us to identify some general trends in the reactivity of anticancer metallodrugs with protein targets.

The reaction of the ruthenium(II) complex fac-[Ru(CO)3Cl2(N1-thz)] (I hereafter; thz = 1,3-thiazole) with human β-amyloid peptide 1-28 (Aβ28) and the resulting {Ru(CO)3}2+ peptide adduct was investigated by a variety of biophysical methods. 1H NMR titrations highlighted a selective interaction of {Ru(CO)3}2+ with Aβ28 histidine residues; circular dichroism revealed the occurrence of a substantial conformational rearrangement of Aβ28; electrospray ionization mass spectrometry (ESI-MS) suggested a prevalent 1:1 metal/peptide stoichiometry and disclosed the nature of peptide-bound metallic fragments. Notably, very similar ESI-MS results were obtained when I was reacted with Aβ42. The implications of the above findings for a possible use of ruthenium compounds in Alzheimer’s disease are discussed.

We have recently shown that a group of structurally diverse gold compounds are highly cytotoxic toward a panel of 36 human tumor cell lines through a variety of biochemical mechanisms. A classic proteomic approach is exploited here to gain deeper insight into those mechanisms. This investigation is focused on Auoxo6, a novel binuclear gold(III) complex, and auranofin, a clinically established gold(I) antiarthritic drug. First, the 72-h cytotoxicity profiles of Auoxo6 and auranofin were determined against A2780 human ovarian carcinoma cells. Subsequently, protein extraction from gold-treated A2780 cells sensitive to cisplatin and 2D gel electrophoresis separation were carried out according to established procedures. Notably, both metallodrugs caused relatively modest changes in protein expression in comparison with controls as only 11 out of approximately 1,300 monitored spots showed appreciable quantitative changes. Very remarkably, six altered proteins were in common between the two treatments. Eight altered proteins were identified by mass spectrometry; among them was ezrin, a protein associated with the cytoskeleton and involved in apoptosis. Interestingly, two altered proteins, i.e., peroxiredoxins 1 and 6, are known to play crucial roles in the cell redox metabolism. Increased cleavage of heterogeneous ribonucleoprotein H was also evidenced, consistent with caspase 3 activation. Overall, the results of the present proteomic study point out that the mode of action of Auoxo6 is strictly related to that of auranofin, that the induced changes in protein expression are limited and selective, that both gold compounds trigger caspase 3 activation and apoptosis, and that a few affected proteins are primarily involved in cell redox homeostasis.

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.

The tetrapeptide Ac-Gly-[Cys-Sec]-Gly-NH2, reproducing the C-terminal motif of the selenoenzyme thioredoxin reductase, was designed and synthesized, and its reactions with a few medicinally relevant gold(I,III) compounds investigated by ESI-MS. Remarkably, the main reaction products could be unambiguously identified providing valuable insight into the likely mechanisms of enzyme inhibition by gold compounds.

The reactivity of cis-diamminediiodidoplatinum(II), cis-PtI2(NH3)2, the iodo analogue of cisplatin, with hen egg white lysozyme (HEWL) was investigated by electrospray ionization mass spectrometry and X-ray crystallography. Interestingly, the study compound forms a stable 1:1 protein adduct for which the crystal structure was solved at 1.99 Å resolution. In this adduct, the PtII center, upon release of one ammonia ligand, selectively coordinates to the imidazole of His15. Both iodide ligands remain bound to platinum, with this being a highly peculiar and unexpected feature. Notably, two equivalent modes of PtII binding are possible that differ only in the location of I atoms with respect to ND1 of His15. The structure of the adduct was compared with that of HEWL–cisplatin, previously described; differences are stressed and their important mechanistic implications discussed.

Abstract A series of organometallic Au I 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 Au I 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.

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.

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.

Au(PEt3)I (AF-I hereafter), the iodide analogue of the FDA-approved drug auranofin (AF hereafter), is a promising anticancer agent that produces its pharmacological effects through interaction with non-genomic targets such as the thioredoxin reductase system. AF-I is endowed with a very favorable biochemical profile showing potent in vitro cytotoxic activity against several cancer types including ovarian and colorectal. Remarkably, in a recent publication, some of us reported that AF-I induces an almost complete and rapid remission in an orthotopic in vivo mouse model of ovarian cancer. The cytotoxic potency does not bring about highly severe side effects making AF-I very well tolerated even for higher doses than the pharmacologically active ones. All these promising features led us to expand our studies on the mechanistic aspects underlying the antitumor activity of AF-I. We report here on an integrated experimental and theoretical study on the reactivity of AF-I, in comparison with auranofin, toward relevant aminoacidic residues or their molecular models. Results point out that the replacement of the thiosugar moiety with iodide affects significantly the overall reactivity toward the amino acid residues histidine, cysteine, methionine and selenocysteine. Altogether, the obtained results contribute to shed light into the enhanced antitumoral activity of AF-I compared with AF.

The discovery of the antineoplastic properties of cisplatin in 1965 by Rosenberg and co-workers, originated a renewed attraction for metal complexes for medicinal applications. Indeed, after its first outstanding clinical results, chemists involved in the metal complexes research field readily started to study safer and more effective alternatives to cisplatin itself. In this frame, after decades of intensive research mainly focused on classical Pt(II) compounds, also platinum(IV)-based compounds have been taken into account. The reason for this interest is that, despite the kinetic inertness of Pt(IV) compounds, they can undergo to “in-cell” reduction process which is able to activate the platinum centre through the generation of the more reactive Pt(II) counterpart. This kind of approach might offer some relevant advantages, such as the reduction of severe side effects associated with Pt(II) off-target reactions. Indeed, the lower redox potential of cancer cells with respect to the healthy ones is able to trigger the Pt(IV) reduction into the biologically active counterpart. In this review, we summarized the most recent goals achieved in the field of so-called Pt(IV) prodrugs.

The solution behaviour of some novel organogold(III) compounds was investigated, and their cytotoxic properties evaluated against a few human tumour cell lines (A2780/S, A2780/R, MCF7, HT29 and A549). Specifically, the following compounds were considered: [Au(bipydmb-H)(2,6-xylidine-H)][PF6] (AuXyl) and [Au(bipydmb-H)(p-toluidine-H)][PF6] (AuTol) (in which bipydmb = 6-(1,1-dimethylbenzyl)-2,2′-bipyridine), [Au(pydmb-H)(AcO)2] (AuPyAcO) (in which pydmb = 2-(1,1-dimethylbenzyl)-pyridine) and [Au(pzPh-H)Cl3]K (AuPzCl) (in which pzPh = 1-phenylpyrazole). The solution chemistry of these compounds, under physiological-like conditions, was investigated through UV–vis absorption and 1H NMR spectroscopies. Significant cytotoxic effects in vitro were observed in selected cases.

The reactions with hemoglobin of artemisinin and of its parent compounds, sodium artesunate and dihydroartemisinin, were investigated by visible absorption spectroscopy under standard solution conditions (50 mM phosphate buffer, pH 7, 37 °C). Notably, these antimalarial drugs were found to react with hemoglobin (i.e., ferrous heme), but not with methemoglobin (i.e., ferric heme). The reaction selectively occurs at the heme sites and consists of the progressive, slow decay of the Soret band, as a consequence of heme alkylation and subsequent loss of π electron delocalization. For the various tested compounds the process reaches completion within ∼30–70 h. Additional experiments were carried out upon adopting the solution conditions described by Meunier et al. and by Kannan et al. in their recent studies. Some reactivity of artemisinin with methemoglobin was indeed detected after addition of 50% v/v acetonitrile, most likely as a consequence of extensive protein unfolding. A unified description for the reactions of artemisinins with hemoglobin is given.

The reaction of metallothionein-2 (MT-2) with the organometallic antitumour compound [Ru(η(6)-p-cymene)Cl(2)(pta)], RAPTA-C, was investigated using ESI MS and ICP AES. The studies were performed in comparison to cisplatin and significant differences in the binding of the two complexes were observed. RAPTA-C forms monoadducts with MT-2, at variance with cisplatin, that has been observed to form up to four adducts. These data, combined with ICP AES analysis, show that binding of both RAPTA-C and cisplatin to MT-2 requires the displacement of an equivalent amount of zinc, suggesting that Cys residues are the target binding sites for the two metallodrugs. The competitive binding of RAPTA-C and cisplatin towards a mixture of ubiquitin (Ub) and MT-2 was also studied, showing that MT-2 can abstract RAPTA-C from Ub more efficiently than it can abstract cisplatin. The mechanistic implications of these results are discussed.

The reaction of aqueous cis-[Pt(NH3)2(H2O)2](NO3)2 with Na+HMEL− (H2MEL, meloxicam, 4-hydroxy-2-methyl-N-(5-methyl-1,3-thiazol-2-yl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide), and Na+HISO− (H2ISO, isoxicam, 4-hydroxy-2-methyl-N-(5-methylisoxazol-3-yl)-2H-1,2-benzothiazine-3-carboxamide-1,1-dioxide) at pH 7 produced micro-crystalline cis-[Pt(NH3)2(N1′-HMEL)2], 5 and cis-[Pt(NH3)2(N1′-HISO)2], 6. The X-ray diffraction structure of 5 shows two HMEL− anions donating through the thiazole nitrogen atoms and adopting a head-to-tail (HT) conformation. The 1H NMR spectrum for 5 from DMSO-d6 shows inertness of the complex up to at least 24 h. Delivery studies for 5 and 6 from vinyl hydrogel based on l-phenylalanine (pH 6.5, 25 °C) show that concentrations of complexes ranging between 2.5 and 5 μM can be reached after a day. Compounds 5 and 6 show strong anti-proliferative effects on CH1 cells (ovarian carcinoma, human) in vitro, IC50 values being 0.60 and 0.37 μM, respectively (0.16 μM for reference, cis-diamminodichloridoplatinum(II), cisplatin). ESI-MS measurements clearly documented that both 5 and 6 form adducts with the three model proteins ubiquitin (UBI), cytochrome c (CYT C) and superoxide dismutase (SOD), the HISO− complex being significantly more effective than the HMEL− one. Density functional methods help in finding rationale for the easiest dissociation of Pt–H2ISO/HISO bonds when compared to the Pt–N1′–H2MEL/N1′–HMEL linkages.

Interactions of therapeutic drugs containing metals with proteins are known to exert a great impact on the mode of action of these compounds, including drug metabolism, delivery, cell processing, and targeting. Modern analytical techniques applied to proteomic studies of metallodrugs may improve our understanding of accompanying biochemical processes, which is essential for the efficiency of treatment, the proper dosing of established metal-based cancerostatic agents, and the design and development of new drugs. Such methods basically rely on the application of mass spectrometry (or a few alternative detection techniques) for species identification, characterization, quantification, and measuring the binding parameters, directly or after separation of free parent drug and protein-bound drug fractions, using the principles of electrophoresis, chromatography or ultrafiltration. This review focuses on the development and recent advances in the field of "metallodrug proteomics" from the implementation of advanced analytical methodologies. Also addressed are emerging issues of metallodrug binding toward cellular protein targets and within real-world biological samples.

The anticancer behaviour of Ru arene complexes can be tuned by an appropriate choice of the site and linkage of the bioactive group to the phosphane ligand.

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. 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.

PtI₂(DACH) has been prepared and tested. PtI₂(DACH) shows different chemical and biological features than oxaliplatin, manifesting cellular effects nearly comparable to those of parent drug in three cellular lines of CRC.

Solution and solid-state data give a quite clear picture for a bis carbene gold(<sc>i</sc>) complex having perspective anticancer properties.

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.

During the last 50 years, formation of the highly chromogenic copper cuprizone complex has been exploited for spectrophotometric determinations of copper although the precise chemical nature of the resulting species has never been ascertained; we eventually show here, in contrast to current opinion, that copper cuprizone is a copper(III) complex.

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 reaction of bovine serum albumin (BSA) with [trans-RuCl4(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 L3-edges. Ruthenium K and L3-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 interactions of anticancer metallodrugs with proteins are attracting a growing interest in the current literature because of their relevant pharmacological and toxicological consequences. To understand in more depth the nature of those interactions, we have investigated the reactions of four anticancer platinum(II) iminoether complexes, namely, trans- and cis-EE (trans- and cis-[PtCl2{(E)-HNC(OCH3)CH3}2], respectively) and trans- and cis-Z (trans- and cis-[PtCl2(NH3){(Z)-HNC(OCH3)CH3}], respectively), with horse heart cytochrome c (cyt c). Our investigation was performed using mainly electrospray ionization mass spectrometry (ESI MS) but was also supported by NMR, inductively coupled plasma optical emission spectroscopy (ICP OES), and absorption electronic spectroscopy. ESI MS spectra clearly revealed the formation of a variety of platinum−protein adducts predominantly corresponding to monoplatinated cyt c species. From a careful analysis of the major ESI MS peaks, specific information on the nature of the protein-bound metallic fragments and on the underlying metallodrug−cyt c reactions was gained for the various cases. We found that trans-EE produces a major cyt c adduct (12 667 Da) that is different from that produced by either cis-EE or by trans-Z and cis-Z (12 626 Da). In particular, occurrence of extensive hydrolysis/aminolysis (the latter fostered by ammonium carbonate buffer) of the iminoether ligands and formation of the corresponding amides/amidines has been unambiguously documented. The reactivity of the iminoether ligands is greatly enhanced by the presence of cyt c as inferred from comparative NMR solution studies. Additional ESI MS measurements recorded on enzymatically cleaved samples of platinated cyt c adducts, together with NMR investigation of the cyt c/trans-EE adduct, strongly suggest that protein platination primarily occurs at Met 65. The biological and pharmacological implications of the described protein platination processes are discussed.

The reactions of human β-amyloid peptide 1-28 (Aβ28) with Al(III) and Fe(III) ions were investigated by (1)H NMR and electrospray ionization mass spectrometry (ESI-MS) under pH conditions close to physiological ones. (1)H NMR titrations, performed in the 5.3-8.0 pH range, revealed that no measurable amounts of Aβ28-Al(III) or Aβ28-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 Aβ28, 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.

ChemMedChemVolume 5, Issue 12 p. 1989-1994 Communication The X-ray Structure of the Adduct between NAMI-A and Carbonic Anhydrase Provides Insights into the Reactivity of this Metallodrug with Proteins Dr. Angela Casini, Corresponding Author Dr. Angela Casini [email protected] Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland), Fax: (+41) 216-939-865 Angela Casini, Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland), Fax: (+41) 216-939-865 Claudia Temperini, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Luigi Messori, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this authorDr. Claudia Temperini, Corresponding Author Dr. Claudia Temperini [email protected] University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Angela Casini, Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland), Fax: (+41) 216-939-865 Claudia Temperini, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Luigi Messori, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this authorDr. Chiara Gabbiani, Dr. Chiara Gabbiani University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this authorProf. Claudiu T. Supuran, Prof. Claudiu T. Supuran University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this authorProf. Luigi Messori, Corresponding Author Prof. Luigi Messori [email protected] University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Angela Casini, Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland), Fax: (+41) 216-939-865 Claudia Temperini, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Luigi Messori, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this author Dr. Angela Casini, Corresponding Author Dr. Angela Casini [email protected] Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland), Fax: (+41) 216-939-865 Angela Casini, Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland), Fax: (+41) 216-939-865 Claudia Temperini, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Luigi Messori, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this authorDr. Claudia Temperini, Corresponding Author Dr. Claudia Temperini [email protected] University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Angela Casini, Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland), Fax: (+41) 216-939-865 Claudia Temperini, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Luigi Messori, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this authorDr. Chiara Gabbiani, Dr. Chiara Gabbiani University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this authorProf. Claudiu T. Supuran, Prof. Claudiu T. Supuran University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this authorProf. Luigi Messori, Corresponding Author Prof. Luigi Messori [email protected] University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Angela Casini, Institut des Sciences et Ingénierie Chimiques, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne (Switzerland), Fax: (+41) 216-939-865 Claudia Temperini, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385 Luigi Messori, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Firenze (Italy), Fax: (+39) 055-457-3385Search for more papers by this author First published: 07 October 2010 https://doi.org/10.1002/cmdc.201000331Citations: 38Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. 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Platinum-based chemotherapy is the primary treatment for human ovarian cancer. Overcoming platinum resistance has become a critical issue in the current chemotherapeutic strategies of ovarian cancer as drug resistance is the main reason for treatment failure. Cytotoxic gold compounds hold great promise to reach this goal; however, their modes of action are still largely unknown. To shed light on the underlying molecular mechanisms, we performed 2-DE and MS analysis to identify differential protein expression in a cisplatin-resistant human ovarian cancer cell line (A2780/R) following treatment with two representative gold compounds, namely Auranofin and Auoxo6. It is shown that Auranofin mainly acts by altering the expression of Proteasome proteins while Auoxo6 mostly modifies proteins related to mRNA splicing, trafficking and stability. We also found that Thioredoxin-like protein 1 expression is greatly reduced after treatment with both gold compounds. These results are highly indicative of the likely sites of action of the two tested gold drugs and of the affected cellular functions. The implications of the obtained results are thoroughly discussed in the frame of current knowledge on cytotoxic gold agents.

A series of new gold(I) and gold(III) complexes based on the saccharinate (sac) ligand, namely M[Au(sac)2] (with M being Na+, K+ or NH4+), [(PTA)Au(sac)], K[Au(sac)3Cl] and Na[Au(sac)4], were synthesized and characterized, and some aspects of their biological profile investigated. Spectrophotometric analysis revealed that these gold compounds, upon dissolution in aqueous media, at physiological pH, manifest a rather favourable balance between stability and reactivity. Their reactions with the model proteins cytochrome c and lysozyme were monitored by mass spectrometry to predict their likely interactions with protein targets. In the case of disaccharinato gold(I) complexes, cytochrome c adducts bearing four coordinated gold(I) ions were preferentially formed in high yield. In contrast, [(PTA)Au(sac)] (PTA = 1,3,5-triaza-7-phosphaadamantane) turned out to be poorly effective, only producing a mono-metalated adduct in very low amount. In turn, the gold(III) saccharinate derivatives were less reactive than their gold(I) analogues: K[Au(sac)3Cl] and Na[Au(sac)4] caused moderate protein metalation, again with evidence of formation of tetragold adducts. Finally, the above mentioned gold compounds were challenged against the reference human tumor cell line A2780S and its cisplatin resistant subline A2780R and their respective cytotoxic profiles determined. [(PTA)Au(sac)] turned out to be highly cytotoxic whereas moderate cytotoxicities were observed for the gold(III) complexes and only modest activities for disaccharinato gold(I) complexes. The implications of these results are thoroughly discussed in the light of current knowledge on gold based drugs.

Six diiodido–diamine platinum(II) complexes, either cis or trans configured, were prepared, differing only in the nature of the amine ligand (isopropylamine, dimethylamine, or methylamine), and their antiproliferative properties were evaluated against a panel of human tumor cell lines. Both series of complexes manifested pronounced cytotoxic effects, with the trans isomers being, generally, more effective than their cis counterparts. Cell cycle analysis revealed different modes of action for these new Pt(II) complexes with respect to cisplatin. The reactivity of these platinum compounds with a number of biomolecules, including cytochrome c, two sulfur containing modified amino acids, 9-ethylguanine, and a single strand oligonucleotide, was analyzed in depth by mass spectrometry and NMR spectroscopy. Interestingly, significant differences in the reactivity of the investigated compounds toward the various model biomolecules were observed: in particular we observed that trans complexes preferentially release their iodide ligands upon biomolecule binding, while the cis isomers may release the amine ligands with retention of iodides. Such differences in reactivity may have important mechanistic implications and a relevant impact on the respective pharmacological profiles.

DIGE (difference in gel electrophoresis) proteomics is exploited here to gain insight into the molecular mechanisms of two established ruthenium-based antimetastatic agents, namely trans-[tetrachloro (DMSO) (imidazole)ruthenate(III)] (NAMI-A) and [Ru(η6-toluene)Cl2(PTA)] (RAPTA-T), where PTA is 1,3,5-triaza-7-phosphaadamantane. Following 24 h exposure of A2780/S human ovarian carcinoma cells to pharmacologically relevant concentrations of either ruthenium compound, 2D-DIGE proteomic analysis evidenced only few differentially expressed proteins with respect to controls. Successive mass spectrometry measurements, MALDI-TOF (matrix assisted laser desorption ionization-time of flight) or LC–ESI/MS-MS (liquid chromatography–electrospray ionization/multi-stage mass spectrometry), allowed identification of most altered protein spots, some of which were associated to perturbations in specific cellular functions. Direct insight into the cellular effects of the investigated metallodrugs is thus achieved. Notably, the patterns of protein alterations induced by NAMI-A and RAPTA-T are quite similar to each other while being deeply different from those of cisplatin. To the best of our knowledge this is the first proteomic study on human cancer cells investigating responses to antimetastatic ruthenium drugs. The key role of new “omic” approaches for deciphering the elusive and complex biochemical mechanisms through which anticancer metallodrugs produce their pharmacological effects is further documented.

Cisplatin and its analogues are first-line chemotherapeutic agents for the treatment of numerous human cancers. A major inconvenience in their clinical use is their strong tendency to link to sulfur compounds, especially in kidney, ultimately leading to severe nephrotoxicity. To overcome this drawback we prepared a variety of platinum complexes with sulfur ligands and analyzed their biological profiles. Here, a series of six platinum(II) compounds bearing a conserved O,S binding moiety have been synthesized and characterized as experimental anticancer agents. The six compounds differ in the nature of the O,S bidentate β-hydroxydithiocinnamic alkyl ester ligand where both the substituents on the aromatic ring and the length of the alkyl chain may be varied. The two remaining coordination positions at the square-planar platinum(II) center are occupied by a chloride ion and a DMSO molecule. These novel platinum compounds showed an acceptable solubility profile in mixed DMSO-buffer solutions and an appreciable stability at physiological pH as judged from analysis of their time-course UV-visible absorption spectra. Their anti-proliferative and pro-apoptotic activities were tested against the cisplatin-resistant lung cancer cell line A549. Assays revealed significant effects of the sample drugs at low concentrations (in the μmolar range); initial structure-activity-relationships are proposed. The activity of the apoptosis-promoting protein caspase 3/7 was determined; results proved that these novel platinum compounds, under the chosen experimental conditions, preferentially induce apoptosis over necrosis. Reactions with the model proteins cytochrome c, lysozyme and albumin were studied by ESI MS and ICP-OES to gain preliminary mechanistic information. The tested compounds turned out to metalate the mentioned proteins to a large extent. In view of the obtained results these novel platinum complexes qualify themselves as promising cytotoxic agents and merit, in our opinion, a deeper pharmacological evaluation as prospective anticancer agents.

Gold(III) compounds form a family of promising cytotoxic and potentially anticancer agents that are currently undergoing intense preclinical investigations. Four recently synthesized and characterized gold(III) derivatives of 2-substituted pyridines are evaluated here for their biological and pharmacological behavior. These include two cationic adducts with 2-pyridinyl-oxazolines, [Au(pyoxR)Cl2][PF6], [pyoxR = (S)-4-benzyl-2-(pyridin-2-yl)-4,5-dihydrooxazole, I; (S)-4-iso-propyl-2-(pyridin-2-yl)-4,5-dihydrooxazole, II] and two neutral complexes [Au(N,N′OH)Cl2], III, and [Au(N,N′,O)Cl], IV, containing the deprotonated ligand N-(1-hydroxy-3-iso-propyl-2-yl)pyridine-2-carboxamide, N,N′H,OH, resulting from ring opening of bound pyoxR ligand of complex II by hydroxide ions. The solution behavior of these compounds was analyzed. These behave as classical prodrugs: activation of the metal center typically takes place through release of the labile chloride ligands while the rest of the molecule is not altered; alternatively, activation may occur through gold(III) reduction. All compounds react eagerly with the model protein cyt c leading to extensive protein metalation. ESI MS experiments revealed details of gold–cyt c interactions and allowed us to establish the nature of protein bound metal containing fragments. The different behavior displayed by I and II compared to III and IV is highlighted. Remarkable cytotoxic properties, against the reference human ovarian carcinoma cell lines A2780/S and A2780/R were disclosed for all tested compounds with IC50 values ranging from 1.43 to 6.18 μM in the sensitive cell line and from 1.59 to 10.86 μM in the resistant one. The common ability of these compounds to overcome cisplatin resistance is highlighted. The obtained results are thoroughly discussed in the frame of current knowledge on cytotoxic gold compounds.

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.

Quite surprisingly, cisplatin and cis-[PtI2(NH3)2] were found to manifest significant differences in their reactions with the model protein lysozyme. We decided to explore whether these differences recur when reacting these two Pt compounds with other proteins. Notably, ESI-MS measurements carried out on cytochrome c nicely confirmed the reaction pattern observed for lysozyme. This prompted us to exploit a computational DFT approach to disclose the molecular basis of such behavior. We analyzed comparatively the reactions of cis-[PtCl2(NH3)2] and cis-[PtI2(NH3)2] with appropriate molecular models (Ls) of the sidechains of relevant aminoacids. We found that when Pt(II) complexes are reacted with sulfur ligands both quickly lose their halide ligands and then the resulting cis-[Pt(L)2(NH3)2] species loses ammonia upon reaction with a ligand excess. In the case of imidazole, again cis-[PtCl2(NH3)2] and cis-[PtI2(NH3)2] quickly lose their halide ligands but the resulting cis-[Pt(L)2(NH3)2] species does not lose ammonia by reaction with excess imidazole. These results imply that the two platinum complexes manifest a significantly different behavior in their reaction with representative small molecules in agreement with what observed in the case of model proteins. It follows that the protein itself must play a crucial role in triggering the peculiar reactivity of cis-[PtI2(NH3)2] and in governing the nature of the formed protein adducts. The probable reasons for the observed behavior are critically commented and discussed.

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.

Novel and surprising biological properties were disclosed for the platinum(II) complex cis-diiodidodiisopropylamineplatinum(II).Remarkably, this new platinum(II) complex manifests pronounced antiproliferative properties in vitro, in some cases superior to those of cisplatin.A peculiar reactivity with the model protein cytochrome c was indeed highlighted based on the loss of amine ligands and retention of iodides.

Based on ESI-MS measurements, we show here that some representative cytotoxic gold(III) compounds produce stable adducts upon reaction with the copper chaperone Atox-1; notably, such adducts contain gold in the oxidation state +1. These findings are of interest to understand the intracellular metabolism of medicinal gold species and to develop new potent inhibitors of the copper trafficking system.

Gold compounds form an interesting class of antiproliferative agents of potential pharmacological use in cancer treatment. Indeed, a number of gold compounds, either gold(III) or gold(I), were recently described and characterised that manifested remarkable cytotoxic properties in vitro against cultured cancer cells; for some of them encouraging in vivo results were also reported toward a few relevant animal models of cancer. The molecular mechanisms through which gold compounds exert their biological effects are still largely unknown and the subject of intense investigations. Recent studies point out that the modes of action of cytotoxic gold compounds are essentially DNA-independent and cisplatin-unrelated, relying -most likely- on gold interactions with a variety of protein targets. Notably, a few cellular proteins playing relevant functional roles were proposed to represent effective targets for cytotoxic gold compounds but these hypotheses need adequate validation. The state of the art of this research area and the perspectives for future studies are herein critically analysed and discussed. Keywords: Gold compounds, cancer, proteins, metal-protein adducts, phosphine complexes, antiproliferative properties, dithiocarbamate complexes, thiosugar ligand, X-ray diffraction, cytochrome c

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 Au2Phen. 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 Au2Phen-treated cells. In conclusion, we performed a comprehensive analysis of proteins regulated by AuL12 and Au2Phen, providing a useful insight into their mechanisms of action.

Abstract Au III 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 Au III ‐NHC complexes by direct oxidation with radioactive [ 124 I]I 2 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 Au III ‐to‐Au I reduction to achieve full anticancer activity, in vivo studies reveal that a fraction of the Au III ‐NHC prodrug is not immediately reduced after administration but able to reach the major organs before metabolic activation.

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.

A panel of four novel gold(I) complexes, inspired by the clinically established gold drug auranofin (1-Thio-β-D-glucopyranosatotriethylphosphine gold-2,3,4,6-tetraacetate), was prepared and characterized. All these compounds feature the replacement of the triethylphosphine ligand of the parent compound auranofin with a trimethylphosphite ligand. The linear coordination around the gold(I) center is completed by Cl-, Br-, I- or by the thioglucose tetraacetate ligand (SAtg). The in-solution behavior of these gold compounds as well as their interactions with some representative model proteins were comparatively analyzed through 31PNMR and ESI-MS measurements. Notably, all panel compounds turned out to be stable in aqueous media, but significant differences with respect to auranofin were disclosed in their interactions with a few leading proteins. In addition, the cytotoxic effects produced by the panel compounds toward A2780, A2780R and SKOV-3 ovarian cancer cells were quantitated and found to be in the low micromolar range, since the IC50 of all compounds was found to be between 1 μM and 10 μM. Notably, these novel gold complexes showed large and similar inhibition capabilities towards the key enzyme thioredoxin reductase, again comparable to those of auranofin. The implications of these results for the discovery of new and effective gold-based anticancer agents are discussed.

Cationic liposomes give rise to stable complexes with DNA molecules (lipoplexes) that are of great interest for gene delivery applications. In particular, liposomes made up by a cationic lipid (DOTAP or DC-Chol) and a zwitterionic lipid (DOPE), produce stable adducts with single and double-stranded DNA oligonucleotides. Formation of these lipoplexes has been further addressed here by circular dichroism spectroscopy (CD) and by other independent biophysical methods. Titration of DNA oligonucleotides with cationic liposomes resulted into significant modifications of their circular dichroic bands. Such spectral modifications were ascribed to progressive DNA condensation and loss of native conformation, as a consequence of the electrostatic interactions taking place between the phosphate groups of DNA and the positively charged head groups of cationic lipids. In all cases, the loss of the CD feature characteristic of the native DNA conformation closely matched the inflection point of Zeta potential profiles. The resulting adducts showed peculiar and non-canonical CD spectra, while exhibiting appreciable stability at physiological pH.

Three novel cyclic tetrapeptides, containing either l- or d-histidine residues and either Lys or Asp side chains, namely c(HGd-HK) (1), cHGHD (2) and c(HGd-HD) (3), were designed, synthesized, characterized and tested as potential copper(II) ligands. Their pH dependent copper(II) binding properties were analysed in depth by a number of potentiometric and spectroscopic determinations. A rather exhaustive description of the species existing in solution has emerged for each copper(II)/oligopeptide system; solution structures for the individual species are proposed. The specific role of the various side chains in the overall metal coordination process is discussed in comparison with the case of Cu(II)-c(HGHK), previously reported. Data obtained in this study highlight the strong impact of the d-His residue on the metal binding abilities of these cyclic peptides. Remarkably, the cyclic tetrapeptides containing two l-His residues are able to form, at physiologically relevant pH values, a characteristic chromophore where the mononuclear copper(II) centre is simultaneously coordinated by two imidazole nitrogens and two amidic nitrogens of the tetrazadodecane ring. This latter type of copper(II) chromophore has been carefully modelled by computational methods. The potentialities of the applied experimental strategy are stressed.

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.

A variegate group of metallodrugs was evaluated in vitro for antimalarial activity through the pLDH test. The panel comprised one mononuclear gold(III) complex, (Aubipy), three dinuclear gold(III) compounds (Auoxo4, Auoxo5 and Auoxo6), three ruthenium(III) complexes (NAMI A, PMRU20, PMRU27), one ruthenium(II) complex (PMRU52), one bismuth(III) compound (Bismuth citrate), antimony trichloride (SbCl(3)) and arsenic trioxide (As(2)O(3)). This panel, although relatively small, was built up in such a way to include a variety of metal centers, structural motifs and metal coordination environments. In general, the tested compounds turned out to contrast effectively Plasmodium falciparum growth in vitro. In two cases, i.e. NAMI A and antimony trichloride, IC(50) values in the high nanomolar range were measured. Notably, the antiplasmodial effects appear not to be correlated to in vitro anticancer properties. The mechanistic and pharmacological implications of the obtained results are discussed.

Twelve Pt(II) complexes with cis-PtP(2)S(2) pharmacophores (where P(2) refers to two monodentate or one bidentate phosphane ligand and S(2) is a dithiolato ligand) were prepared, characterized and evaluated as potential antiproliferative agents. The various compounds were first studied from the structural point of view; afterward, their solubility properties as well as their solution behaviour were analyzed in detail. Antiproliferative properties were specifically evaluated against A2780 human ovarian carcinoma cells, either resistant or sensitive to cisplatin. For comparison purposes similar studies were carried out on four parent cis-dichloro bisphosphane Pt(II)complexes. On the whole, the cis-PtP(2)S(2) compounds displayed significant antiproliferative properties while the cis-PtP(2)Cl(2) (cis-dichloro bisphosphane Pt(II)) compounds revealed quite poor biological performances. To gain further insight into the molecular mechanisms of these bisphosphane Pt(II) compounds, the reactions of selected complexes against the model protein cytochrome c were investigated by ESI-MS and their adduct formation explored. A relevant reactivity with cyt c was obtained only for cis-PtP(2)Cl(2) compounds, whereas cis-PtP(2)S(2) compounds turned out to be nearly unreactive. The obtained results are interpreted and discussed in the frame of the current knowledge of anticancer platinum compounds and their structure-activity-relationships. The observation of appreciable antiproliferative effects for the relatively inert cis-PtP(2)S(2) compounds strongly suggests that these compounds will undergo specific activation within the cellular environment.

Abstract Thioredoxin reductase (TrxR) is overexpressed in cancer cells and is therefore a putative cancer target. Inhibition of this enzyme is considered an important strategy for the development of new chemotherapeutic agents with a specific mechanism of action. Organotin compounds have been described as experimental antitumor agents, yet their mechanism of action remains largely unknown. Based on the outcome of a virtual screening study, various di‐ and tri‐ n ‐butyltin(IV) carboxylates were synthesized, and their biological properties were evaluated. All synthesized compounds were able to inhibit TrxR selectively within the micromolar range and showed potent antitumor activity against HT‐29 and MCF‐7 cancer cell lines. Moreover, tin(IV) organometallics were found to strongly induce apoptosis in the BJAB lymphoma cell line. Mass spectrometry and atomic absorption spectroscopy experiments revealed metal binding to proteins, and efficient cellular uptake was observed using a di‐ n ‐butyltin(IV) complex as an example.

Two dinuclear oxo-bridged organogold(iii) compounds, namely [(N,N,C)2Au2(μ-O)][PF6]2 (with N,N,CH = 6-(1-methylbenzyl)-2,2′-bipyridine, Au22O1; or 6-(1,1-dimethylbenzyl)-2,2′-bipyridine, Au22O2), were previously prepared and characterised. Their solution chemistry under physiological-like conditions has been investigated here as well as their in vitro antiproliferative properties. Notably, these compounds reveal a marked redox stability even in the presence of effective biological reductants such as ascorbic acid and glutathione. The two dinuclear gold(iii) compounds were evaluated for cytotoxic actions against a representative panel of 12 human tumor cell lines, in comparison to respective mononuclear parent compounds [(N,N,C)AuOH][PF6], and appreciable biological activity could be highlighted. The reactions of Au22O1 and Au22O2 with a few model proteins were studied and the ability to form metallodrug–protein adducts monitored through ESI MS methods. Typical adducts were identified where the protein is associated to monometallic gold fragments; in these adducts gold remains in the oxidation state +3 and conserves its organic ligand. A direct comparison of the biological profiles of these binuclear organogold(iii) compounds with those previously reported for a series of dinuclear oxo-bridged complexes [(N,N)2Au2(μ-O)2][PF6]2 (N,N = 6(6′)-substituted 2,2′-bipyridines) named Auoxo's was carried out. It emerges that the greater cytotoxicity of the latter is mainly due to the greater oxidising power of their gold(iii) centres and to propensity to generate gold(i) species; in contrast, the here described bimetallic organogold(iii) complexes manifest a far higher redox stability in the biological milieu coupled to lower, but still significant, antiproliferative properties. Different molecular mechanisms are thus hypothesised for these two classes of dinuclear gold(iii) agents.

Aubipyc, i.e.[(bipydmb-H)Au(OH)][PF6] (where bipydmb-H = deprotonated 6-(1,1-dimethylbenzyl)-2,2′-bipyridine), Auoxo6, i.e. [(bipy2Me)2Au2(μ-O)2][PF6]2 (where bipy2Me = 6,6′-dimethyl-2,2′-bipyridine) and Au2phen i.e. [(phen2Me)2Au2(μ-O)2][PF6]2 (where phen2Me = 2,9-dimethyl-1,10-phenanthroline), are three representative gold(III) compounds prepared and characterised in our laboratories during the last few years that manifested remarkable anticancer properties in vitro. Herein, the main chemical features of these compounds are summarised. Aubipyc is a mononuclear organogold(III) compound while Auoxo6 and Au2phen are binuclear gold(III) complexes. These compounds manifest a reasonable stability of their gold(III) chromophore in aqueous solutions at physiological pH; yet, a rather different redox behaviour was highlighted as Aubipyc displays high stability toward reduction while both Auoxo6 and Au2phen are rapidly reduced by ascorbic acid and glutathione. The antiproliferative properties of these gold(III) compounds were analysed in detail against a wide panel of human tumour cell lines. Remarkably, Auoxo6 and Au2phen revealed potent and rather similar patterns of antiproliferative actions while Aubipyc turned out to be less effective. For Auoxo6 and Au2phen more detailed biochemical studies are available documenting their effects on the proteome of treated cancer cells. Recent studies described the reactions of these gold compounds with various proteins at the molecular level; adduct formation was clearly documented in a few cases and their nature determined. Preliminary results suggest that these gold compounds may act as strong inhibitors of the selenoenzyme thioredoxin reductase and cause mitochondrial dysfunction. Based on the available in vitro data, these gold compounds look quite promising as prospective anticancer agents. Studies will soon be extended to assess their safety and efficacy in relevant animal models of cancer.

ChemMedChemVolume 8, Issue 10 p. 1634-1637 Communication Metal-Based Compounds as Prospective Antileishmanial Agents: Inhibition of Trypanothione Reductase by Selected Gold Complexes Dr. Gianni Colotti, Corresponding Author Dr. Gianni Colotti [email protected] Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy) Gianni Colotti, Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy) Luigi Messori, Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI) (Italy)Search for more papers by this authorDr. Andrea Ilari, Dr. Andrea Ilari Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy)Search for more papers by this authorDr. Annarita Fiorillo, Dr. Annarita Fiorillo Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy)Search for more papers by this authorDr. Paola Baiocco, Dr. Paola Baiocco Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy)Search for more papers by this authorProf. Maria Agostina Cinellu, Prof. Maria Agostina Cinellu Department of Chemistry & Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari (SS) (Italy)Search for more papers by this authorDr. Laura Maiore, Dr. Laura Maiore Department of Chemistry & Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari (SS) (Italy)Search for more papers by this authorFederica Scaletti, Federica Scaletti Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI) (Italy)Search for more papers by this authorDr. Chiara Gabbiani, Dr. Chiara Gabbiani Department of Chemistry & Industrial Chemistry, University of Pisa, Via Risorgimento 35, 56126 Pisa (Italy)Search for more papers by this authorProf. Luigi Messori, Corresponding Author Prof. Luigi Messori [email protected] Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI) (Italy) Gianni Colotti, Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy) Luigi Messori, Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI) (Italy)Search for more papers by this author Dr. Gianni Colotti, Corresponding Author Dr. Gianni Colotti [email protected] Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy) Gianni Colotti, Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy) Luigi Messori, Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI) (Italy)Search for more papers by this authorDr. Andrea Ilari, Dr. Andrea Ilari Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy)Search for more papers by this authorDr. Annarita Fiorillo, Dr. Annarita Fiorillo Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy)Search for more papers by this authorDr. Paola Baiocco, Dr. Paola Baiocco Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy)Search for more papers by this authorProf. Maria Agostina Cinellu, Prof. Maria Agostina Cinellu Department of Chemistry & Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari (SS) (Italy)Search for more papers by this authorDr. Laura Maiore, Dr. Laura Maiore Department of Chemistry & Pharmacy, University of Sassari, Via Vienna 2, 07100 Sassari (SS) (Italy)Search for more papers by this authorFederica Scaletti, Federica Scaletti Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI) (Italy)Search for more papers by this authorDr. Chiara Gabbiani, Dr. Chiara Gabbiani Department of Chemistry & Industrial Chemistry, University of Pisa, Via Risorgimento 35, 56126 Pisa (Italy)Search for more papers by this authorProf. Luigi Messori, Corresponding Author Prof. Luigi Messori [email protected] Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI) (Italy) Gianni Colotti, Institute of Molecular Biology & Pathology; National Research Council (CNR); Institute Pasteur Fondazione Cenci Bolognetti; Department of Biochemical Sciences, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome (Italy) Luigi Messori, Department of Chemistry, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino (FI) (Italy)Search for more papers by this author First published: 23 August 2013 https://doi.org/10.1002/cmdc.201300276Citations: 16Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Graphical Abstract Picking a fight with parasites! Trypanothione reductase (TR) is a validated drug target for the development of antileishmanial agents. 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Six structurally diverse cytotoxic gold compounds are reported to cause profound and differential inhibition of the three main catalytic activities of purified 20S proteasome whilst auranofin, an established gold(I) drug in clinical use, is nearly ineffective. In particular, the gold(I) complex [(pbiH)Au(PPh3)]PF6, turns out to be the most potent inhibitor of all three enzyme activities with sub-micromolar IC50 values. The present results further support the view that proteasome inhibition may play a major – yet not exclusive – role in the cytotoxic actions of gold based anticancer agents.

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.

A number of structurally diverse gold compounds were evaluated as possible inhibitors of Falcipain 2 (Fp2), a cysteine protease from P. falciparum that is a validated target for the development of novel antimalarial drugs. Remarkably, most tested compounds caused pronounced but reversible inhibition of Fp2 with Ki values falling in the micromolar range. Enzyme inhibition is basically ascribed to gold binding to catalytic active site cysteine. The same gold compounds were then tested for their ability to inhibit P. falciparum growth in vitro; important parasite growth inhibition was indeed observed. However, careful analysis of the two sets of data failed to establish any direct correlation between enzyme inhibition and reduction of P. falciparum growth suggesting that Fp2 inhibition represents just one of the various mechanisms through which gold compounds effectively antagonize P. falciparum replication.

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.

A novel gold(I) complex inspired by the known medicinal inorganic compounds auranofin and thimerosal, namely ethylthiosalicylate(triethylphosphine)gold(I) (AFETT hereafter), was synthesized and characterised and its structure was resolved through X-ray diffraction. The solution behavior of AFETT and its interactions with two biologically relevant proteins (i.e. human serum albumin and haemoglobin) and with a synthetic dodecapeptide reproducing the C-terminal portion of thioredoxin reductase were comparatively analyzed through 31P NMR and ESI-MS. Remarkable binding properties toward these biomolecules were disclosed. Moreover, the cytotoxic effects produced by AFETT on two ovarian cancer cell lines (A2780 and A2780 R) and one colorectal cancer cell line (HCT116) were analyzed and found to be strong and nearly superimposable to those of auranofin. Interestingly, for both compounds, the ability to induce downregulation of vimentin expression in A2780 R cells was evidenced. Despite its close similarity to auranofin, AFETT is reported to exhibit some peculiar and distinctive features such as a lower lipophilicity, an increased water solubility and a faster reactivity towards the selected target biomolecules. These differences might confer to AFETT significant pharmaceutical and therapeutic advantages over auranofin itself.

Three gold(I) linear compounds, sharing the general formula [AuI(LPh3)], have been synthesized and characterized. The nature of the ligand has been modified by moving down among some of the elements of group 15, i.e. phosphorus, arsenic and antimony. The structures of derived compounds have been solved through XRD and the reactivity behaviour towards selected biomolecules has been investigated through a multi-technique approach involving NMR, high-resolution mass spectrometry and IR. Moreover, the biological activity of the investigated compounds has been comparatively analyzed through classical methodologies and the disclosed differences are discussed in detail.

The new cyclic tetrapeptide c(HGHK) was synthesised in the solid phase and its complexes with copper(II) were studied in aqueous solution at various pH values by means of potentiometric and spectroscopic methods (UV, EPR, CD). Six mononuclear coordination species were clearly identified within the pH range 3-11. Spectroscopic data strongly suggest sequential formation of N, 2N, 3N and 4N equatorial donor sets around the copper(II) centre from the lowest to the highest pH, involving both imidazole nitrogens and amide nitrogens. A detailed comparison with the copper(II) binding properties of HGHG and Ac-HGHG ligands is also reported.

Keppler-type ruthenium(III) complexes exhibit promising antitumor properties. We report here a study of 2-aminothiazolium[trans-tetrachlorobis(2-aminothiazole)ruthenate(III)], both in the solid state and in solution. The crystal structure has been solved and found to exhibit classical features. Important solvatochromic effects were revealed. Notably, we observed that introduction of an amino group in position 2 greatly accelerates chloride hydrolysis compared to the thiazole analogue; this latter finding may be of interest for a fine-tuning of the reactivity of these novel metallodrugs.

Abstract A novel trans ‐platinum(II) complex bearing one dimethylamine (dma) and one methylamine (ma) ligand, namely trans ‐[PtCl 2 (dma)(ma)], recently synthesised and characterised in our laboratory, displayed relevant antiproliferative properties in vitro, being more active than the parent complex, trans ‐[PtCl 2 (dma)(ipa)], which has isopropylamine (ipa) in place of methylamine. We have analysed comparatively the solution behaviour of these two complexes under various experimental conditions, and investigated their reactivity with horse heart cytochrome c by mass spectrometry, inductively coupled plasma–optical emission spectroscopy (ICP‐OES), 2D [ 1 H, 15 N],[ 1 H, 13 C] HSQC and [ 1 H, 1 H] NOESY NMR. Some important changes that occurred in the [ 1 H, 13 C] HSQC NMR spectrum of cytochrome c treated with trans ‐[PtCl 2 (dma)(ma)] in water, after two days’ incubation, most probably arose from direct platinum coordination to the protein side chain; this was proved conclusively by [ 1 H, 1 H] NOESY NMR and [ 1 H, 15 N] HSQC NMR measurements. Met65 was identified as the primary Pt binding site on cytochrome c . Electrospray mass spectrometry (ESIMS) results provided evidence for extensive platinum–protein adduct formation. A fragment of the [Pt(amine)(amine′)] type was established to be primarily responsible for protein metalation. ICP‐OES analysis revealed that these trans ‐platinum(II) complexes bind preferentially to the serum proteins albumin and transferrin rather than to calf thymus DNA. Pt binding to DNA was found to be far lower than in the case of cisplatin. The implications of the results for the mechanism of action of novel cytotoxic trans ‐platinum complexes are discussed.

From the polar extracts of the leaves of Quercus ilex L., two new proanthocyanidin glycosides, namely afzelechin-(4α→8)-catechin-3-O-β-glucopyranoside (1) and afzelechin-(4α→8)-catechin-3-O-α-rhamnopyranoside (2), were isolated in addition to catechin (3), proanthocyanidin B3 (4), prodelphinidin C (5), dehydrodicatechin A (6), quercetin (7) and six known flavonol glucosides with their acylated derivatives (8–13) and ellagic acid (14). The structures of all isolated compounds were established by spectroscopic means, mainly 1D and 2D NMR, as well as LC/MS and HR-MS spectrometric analyses. The absolute configuration of compound 1 was determined by CD measurements. The proanthocyanidin glycosides are especially interesting, as they possess the sugar in the upper unit of the dimer, which is rare for this type of compounds.

AuL10 ([Au III Cl 2 (dmdt)], dmdt = N , N ‐dimethyldithiocarbamate) and AuL12 ([Au III Br 2 (esdt)], esdt = ethylsarcosine dithiocarbamate) are two extremely promising gold‐based anticancer compounds. Their mechanisms of action are still largely unknown although some specific hypotheses have been proposed. To shed light on their reactivity with probable biological targets, we report here on their interactions with serum albumin and calf‐thymus DNA, taken as model biomolecules. Quite unexpectedly, spectrophotometric investigations revealed substantially different patterns of interaction for these two gold complexes with the aforementioned 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 means of the xCELLigence Real‐Time Cell Analysis system. Measures of impedance by non‐invasive gold microelectrodes at the base of each well indicated that this gold complex inhibits cell proliferation after only 4 h of treatment, producing its cytotoxic effects probably at the membrane level.