We report here on the synthesis of a series of mono- and dinuclear gold(I) complexes exhibiting sulfonated bis(NHC) ligands and novel hydroxylated mono(NHC) Au(I) compounds, which were also examined for their biological activities. Initial cell viability assays show strong antiproliferative activities of the hydroxylated mono(NHC) gold compounds (8 > 9 > 10) against 2008 human ovarian cancer cells even after 1 h incubation. In order to gain insight into the mechanism of biological action of the gold compounds, their effect on the pivotal cellular target seleno-enzyme thioredoxin reductase (TrxR), involved in the maintenance of intracellular redox balance, was investigated in depth. The compounds’ inhibitory effects on TrxR and glutathione reductase (GR) were studied comparatively, using either the pure proteins or cancer cell extracts. The results show a strong and selective inhibitory effect of TrxR, specifically for the hydroxyl-functionalized NHC gold(I) complexes (8–10). Valuable information on the gold compounds’ molecular reactivity with TrxR was gained using the BIAM (biotin-conjugated iodoacetamide) assay and performing competition experiments by mass spectrometry (MS). In good agreement, both techniques suggest the binding affinity of the mono(NHC) Au(I) complexes toward selenols and thiols. Notably, for the first time, bis-carbene formation from mono-carbenes in buffered solution could be observed by MS, which may provide new insights into the speciation mechanisms of bioactive Au(I) NHC complexes. Furthermore, the compounds’ interactions with another relevant in cellulo target, namely telomeric G-quadruplex DNA—a higher-order DNA structure playing key roles in telomere function—was investigated by means of FRET melting assays. The lack of interactions with this type of nucleic acid secondary structure support the idea of selective targeting of the hydrophilic Au(I) NHC compounds toward proteins such as TrxR.

A family of cyclometalated Au(III) complexes featuring a tridentate C^N^C scaffold has been synthesized and characterized. Microwave assisted synthesis of the ligands has also been exploited and optimized. The biological properties of the thus formed compounds have been studied in cancer cells and demonstrate generally moderate antiproliferative effects. Initial mechanistic insights have also been gained on the gold complex [Au(C^N^C)(GluS)] (3), and support the idea that the thioredoxin system may be a target for this family of compounds together with other relevant intracellular thiol-containing molecules.Download high-res image (127KB)Download full-size image

•Metal-based compounds form a promising class of therapeutic agents.•Metallodrugs are pro-drugs which undergo activation processes and speciation in vivo.•Molecular mass spectrometry is a major tool to study the interactions of metallodrugs at a molecular level.•Non-covalent adducts of metal compounds with biomolecules can be detected by mass spectrometry.•Mass spectrometry imaging of metallodrugs in complex samples provides invaluable information on their modes of action.•New chemical rules have to be defined to characterize metallodrugs’ interactions in biological systems.Metal-based compounds form a promising class of therapeutic agents, whose mechanisms of action still need to be elucidated, and that are in general prone to undergo extensive speciation in physiological environment. Thus, determination of the fate of the metal compounds in complex biological systems, contributing to their overall pharmacological and toxicological profiles, is important to develop more rationalised and targeted metal-based drugs. To these aims, a number of spectroscopic and biophysical methods, as well as analytical techniques, are nowadays extensively applied to study the reactivity of metal complexes with different biomolecules (e.g. nucleic acids, proteins, buffer components). Among the various techniques, molecular mass spectrometry (MS) has emerged in the last decade as a major tool to characterise the interactions of metallodrugs at a molecular level.In this review, we present an overview of the information available on the reactivity of various families of therapeutic metallodrugs (mainly anticancer compounds based on Pt, Ru, Au and As) with biomolecules studied by different MS techniques, including high-resolution ESI-, MALDI- and ion mobility-MS among others. Representative examples on the potential of the MS approach to study non-covalent interactions are also discussed. The review is organized to present results obtained on samples with different degrees of complexity, from the interactions of metal compounds with small model nucleophiles (amino acids and nucleobases), model peptides/oligonucleotides, target proteins/nucleic acids, to the analysis of serum, cell extracts and tissue samples. The latter requiring combination of proteomic methods with advanced MS techniques. Correlations between molecular reactivity of metallodrugs and biological activity are hard to establish, but differences in the reactivity of metallodrugs to biomolecules and their different adducts, as revealed by MS methods, may indicate differences in their modes of action. Overall, the knowledge offered by MS methods on metallodrugs speciation is invaluable to establish new rules and define new trends in the periodic table aimed at rationalizing the behavior of metal compounds in complex living systems.Download high-res image (112KB)Download full-size image

Mechanisms of toxicity and cellular transport of anticancer metallodrugs, including platinum-based agents, have not yet been fully elucidated. Here, we studied the toxic effects and accumulation mechanisms of cisplatin in healthy rat kidneys ex vivo, using the Precision Cut Tissue Slices (PCTS) method. In addition, for the first time, we investigated the nephrotoxic effects of an experimental anticancer cyclometallated complex [Au(pyb-H)(PTA)Cl]PF6 (PTA = 1,3,5-triazaphosphaadamantane). The viability of the kidney slices after metallodrug treatment was evaluated by ATP content determination and histomorphology analysis. A concentration dependent decrease in viability of PCKS was observed after exposure to cisplatin or the Au(III) complex, which correlated with the increase in slice content of Pt and Au, respectively. Metal accumulation in kidney slices was analysed by ICP-MS. The involvement of OCTs and MATE transporters in the accumulation of both metal compounds in kidneys was evaluated co-incubating the tissues with cimitedine, inhibitor of OCT and MATE. Studies of mRNA expression of the markers KIM-1, villin, p53 and Bax showed that cisplatin damages proximal tubules, whereas the Au(III) complex preferentially affects the distal tubules. However, no effect of cimetidine on the toxicity or accumulation of cisplatin and the Au(III) complex was observed. The effect of temperature on metallodrug accumulation in kidneys suggests the involvement of a carrier-mediated uptake process, other than OCT2, for cisplatin; while carrier-mediated excretion was suggested in the cases of the Au(III) complex.

The inhibition of water and glycerol permeation via human aquaglyceroporin-3 (AQP3) by gold(III) complexes has been studied by stopped-flow spectroscopy and, for the first time, its mechanism has been described using molecular dynamics (MD), combined with density functional theory (DFT) and electrochemical studies. The obtained MD results showed that the most effective gold-based inhibitor, anchored to Cys40 in AQP3, is able to induce shrinkage of pores preventing glycerol and water permeation. Moreover, the good correlation between the affinity of the Au(III) complex to Cys binding and AQP3 inhibition effects was highlighted, while no influence of the different oxidative character of the complexes could be observed.

Organometallic compounds currently occupy an important place in the field of medicinal inorganic chemistry due to the unique chemical properties of metal coordination compounds. Particularly, metal compounds ligated by N-heterocyclic carbenes (NHC) have shown high potential for biomedical applications as antimicrobial and anticancer agents during the recent 15 years. Although further studies are necessary to validate the modes of action of this family of compounds, a number of biological targets have been identified, including DNA secondary structures. This perspective review aims at providing an overview of the most representative examples of metal NHC complexes reacting with nucleic acids via different binding modes. It is organized according to the type of DNA secondary structure targeted by metal NHCs, highlighting the possible advantages of biomedical applications, including therapy and imaging.

Supramolecular Pd2L4 cages (L = ligand) hold promise as drug delivery systems. With the idea of achieving targeted delivery of the metallacages to tumor cells, the bioconjugation of exo-functionalized self-assembled Pd2L4 cages to peptides following two different approaches is reported for the first time. The obtained bioconjugates were analyzed and identified by high-resolution mass spectrometry.

A series of new heterodinuclear luminescent complexes with two different organic ligands have been synthesized and characterized. A luminescent RuII(polypyridine) moiety and a metal-based anticancer fragment (AuCl, (p-cymene)RuCl2, (p-cymene)OsCl2, (Cp*)RhCl2, or Au-thioglucose) are the two general features of these complexes. All of the bimetallic compounds have been evaluated for their antiproliferative properties in vitro in human cancer cell lines. Only the complexes containing an Au(I) fragment exhibit antiproliferative activity in the range of cisplatin or higher. The photophysical and electrochemical properties of the bimetallic species have been investigated, and fluorescence microscopy experiments have been performed successfully. The most promising bimetallic cytotoxic complexes (i.e., with the Au-thioglucose scaffold) have shown to be easily taken up by cancer cells at 37 °C in the cytoplasm or in specific organelles. Interestingly, experiments repeated at 4 °C showed no uptake of the bimetallic species inside cells, which confirms involvement of active transport processes. To evaluate the role of glucose transporters in the cell uptake of the gold complexes, inhibition of the GluT-1 (glucose transporter isoform with high level of expression in cancer cells) was achieved, showing only scarce influence on the compounds’ uptake. Finally, the observed absence of interactions with nucleic acid model structures suggests that the gold compounds may have different intracellular targets with respect to cisplatin.

The reactivity of three cytotoxic organometallic gold(III) complexes with cyclometalated C,N,N and C,N ligands (either six- or five-membered metallacycles), as well as that of two representative gold(III) complexes with N-donor ligands, with biological nucleophiles has been studied by ESI-MS on ion trap and time-of-flight instruments. Specifically, the gold compounds were reacted with mixtures of nucleophiles containing l-histidine (imine), l-methionine (thioether), l-cysteine (thiol), l-glutamic acid (carboxylic acid), methylseleno-l-cysteine (selenoether), and in situ generated seleno-l-cysteine (selenol) to judge the preference of the gold compounds for binding to selenium-containing amino acid residues. Moreover, the gold compounds’ reactivity was studied with proteins and nucleic acid building blocks. These experiments revealed profound differences between the coordination and organometallic families and even within the family of organometallics, which allowed insights to be gained into the compounds mechanisms of action. In particular, interactions with seleno-l-cysteine appear to reflect well the compounds’ inhibition properties of the seleno-enzyme thioredoxin reductase and to a certain extent their antiproliferative effects in vitro. Therefore, mass spectrometry is successfully applied for linking the molecular reactivity and target preferences of metal-based drug candidates to their biological effects. Finally, this experimental setup is applicable to any other metallodrug that undergoes ligand substitution reactions and/or redox changes as part of its mechanism of action.

Exo-functionalized Pd2L4 cage compounds with attached Ru(II) pyridine complexes were prepared via coordination-driven self-assembly. Unlike most of the previously reported palladium(II) cages, one of these metallocages exhibits an exceptionally high quantum yield of 66%. The presented approach is promising to obtain luminescent coordination complexes for various applications.

Metallosupramolecular systems are promising new tools for pharmaceutical applications. Thus, novel self-assembled Pd(II) coordination cages were synthesized which were exo-functionalized with naphthalene or anthracene groups with the aim to image their fate in cells. The cages were also investigated for their anticancer properties in human lung and ovarian cancer cell lines in vitro. While the observed cytotoxic effects hold promise and the cages resulted to be more effective than cisplatin in both cell lines, fluorescence emission properties were scarce. Therefore, using TD-DFT calculations, fluorescence quenching observed in the naphthalene-based system could be ascribed to a lower probability of a HOMO–LUMO excitation and an emission wavelength outside the visible region. Overall, the reported Pd2L4 cages provide new insights into the chemical–physical properties of this family of supramolecular coordination complexes whose understanding is necessary to achieve their applications in various fields.

•New compounds with Co, Ni, Cu and Zn containing norharmane have been prepared.•The structure of a Cu(II) compound with norharmane and phenanthroline has been determined.•The Cu and Zn compounds show cytotoxic properties in different cancer cell lines.•The cytotoxic properties are comparable to cisplatin.•The activity of the reported coordination complexes follows the Irving-Williams series.New first-row transition-metal compounds with the ligand norharmane (9H-Pyrido[3,4-b]indole; Hnor) are reported. The compounds have the general formula [M(LL)(Hnor)(NO3)2](MeOH)0–1 (M = Co, Ni, Cu, Zn; LL = 2,2′-bipyridyl (bpy), 1,10-phenanthroline (phen)) and have been characterized by physical and analytical methods. X-ray structural analysis revealed that the compound of formula [Cu(phen)(Hnor)(NO3)2], (1) has a distorted 6-coordinated octahedrally-based geometry, with a planar-based [CuN3O] core, where Cu-L varies between 1.99 and 2.04 Å and two weak axial CuO contacts (2.209 and 2.644 Å) from two different nitrates. Based on spectroscopic similarities, the other compounds appear to have the same or very similar coordination geometries. The compounds showed clear cell growth inhibitory effects in two different cancer cell lines in vitro, with the copper and zinc complexes being the most toxic and in fact almost comparable to cisplatin. Flow-cytometry analysis confirmed induction of apoptosis in cancer cells treated with the compounds. Interestingly, co-incubation of the cells with metal complexes and CuCl2 induced an increase in the cytotoxic effects, most likely due to the conversion of the metal compounds in the corresponding, and most active, copper analogues.Copper, Nickel, Cobalt and Zinc compounds with the ligand norharmane and with 2,2′-bipyridyl (bpy) and 1,10-phenanthroline (phen) as co-ligands are presented, including a 3D structure. Evidence for cell growth inhibitory effects in two different cancer cell lines in vitro, are presented.

The pH gating of human AQP3 and its effects on both water and glycerol permeabilities have been fully characterized for the first time using a human red blood cell model (hRBC). For comparison, the effects of pH on the gating of rat AQP3 have also been characterized in yeast. The obtained results highlight similarities as well as differences between the two isoforms. In addition, we investigated the molecular mechanism of hAQP3 pH gating in silico, which may disclose new pathways to AQP regulation by small molecule inhibitors, and therefore may be important for drug development.

Supramolecular Pd2L4 cages (L = ligand) hold promise as drug delivery systems. With the idea of achieving targeted delivery of the metallacages to tumor cells, the bioconjugation of exo-functionalized self-assembled Pd2L4 cages to peptides following two different approaches is reported for the first time. The obtained bioconjugates were analyzed and identified by high-resolution mass spectrometry.

Organometallic compounds currently occupy an important place in the field of medicinal inorganic chemistry due to the unique chemical properties of metal coordination compounds. Particularly, metal compounds ligated by N-heterocyclic carbenes (NHC) have shown high potential for biomedical applications as antimicrobial and anticancer agents during the recent 15 years. Although further studies are necessary to validate the modes of action of this family of compounds, a number of biological targets have been identified, including DNA secondary structures. This perspective review aims at providing an overview of the most representative examples of metal NHC complexes reacting with nucleic acids via different binding modes. It is organized according to the type of DNA secondary structure targeted by metal NHCs, highlighting the possible advantages of biomedical applications, including therapy and imaging.

Metallosupramolecular systems are promising new tools for pharmaceutical applications. Thus, novel self-assembled Pd(II) coordination cages were synthesized which were exo-functionalized with naphthalene or anthracene groups with the aim to image their fate in cells. The cages were also investigated for their anticancer properties in human lung and ovarian cancer cell lines in vitro. While the observed cytotoxic effects hold promise and the cages resulted to be more effective than cisplatin in both cell lines, fluorescence emission properties were scarce. Therefore, using TD-DFT calculations, fluorescence quenching observed in the naphthalene-based system could be ascribed to a lower probability of a HOMO–LUMO excitation and an emission wavelength outside the visible region. Overall, the reported Pd2L4 cages provide new insights into the chemical–physical properties of this family of supramolecular coordination complexes whose understanding is necessary to achieve their applications in various fields.

The inhibition of water and glycerol permeation via human aquaglyceroporin-3 (AQP3) by gold(III) complexes has been studied by stopped-flow spectroscopy and, for the first time, its mechanism has been described using molecular dynamics (MD), combined with density functional theory (DFT) and electrochemical studies. The obtained MD results showed that the most effective gold-based inhibitor, anchored to Cys40 in AQP3, is able to induce shrinkage of pores preventing glycerol and water permeation. Moreover, the good correlation between the affinity of the Au(III) complex to Cys binding and AQP3 inhibition effects was highlighted, while no influence of the different oxidative character of the complexes could be observed.

Exo-functionalized Pd2L4 cage compounds with attached Ru(II) pyridine complexes were prepared via coordination-driven self-assembly. Unlike most of the previously reported palladium(II) cages, one of these metallocages exhibits an exceptionally high quantum yield of 66%. The presented approach is promising to obtain luminescent coordination complexes for various applications.