Elena Michelucci

Exploring metallodrug-protein interactions by mass spectrometry: comparisons between platinum coordination complexes and an organometallic ruthenium compound.

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.

Odorant-Binding Proteins and Chemosensory Proteins in Pheromone Detection and Release in the Silkmoth Bombyx mori

The genome of the silkmoth Bombyx mori contains 44 genes encoding odorant-binding proteins (OBPs) and 20 encoding chemosensory proteins (CSPs). In this work, we used a proteomic approach to investigate the expression of proteins of both classes in the antennae of adults and in the female pheromone glands. The most abundant proteins found in the antennae were the 4 OBPs (PBP, GOBP1, GOBP2, and ABP) and the 2 CSPs (CSP1 and CSP2) previously identified and characterized. In addition, we could detect only 3 additional OBPs and 2 CSPs, with clearly different patterns of expression between the sexes. Particularly interesting, on the other hand, is the relatively large number of binding proteins (1 OBP and 7 CSPs) expressed in the female pheromone glands, some of them not present in the antennae. In the glands, these proteins could be likely involved in the solubilization of pheromonal components and their delivery in the environment.

[Au2(phen2Me)2(μ-O)2](PF6)2, a Novel Dinuclear Gold(III) Complex Showing Excellent Antiproliferative Properties

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.

fac-{Ru(CO)3}2+ Selectively Targets the Histidine Residues of the β-Amyloid Peptide 1-28. Implications for New Alzheimer's Disease Treatments Based on Ruthenium Complexes

The reaction of the ruthenium(II) complex fac-[Ru(CO)(3)Cl(2)(N(1)-thz)] (I hereafter; thz = 1,3-thiazole) with human beta-amyloid peptide 1-28 (Abeta(28)) and the resulting {Ru(CO)(3)}(2+) peptide adduct was investigated by a variety of biophysical methods. (1)H NMR titrations highlighted a selective interaction of {Ru(CO)(3)}(2+) with Abeta(28) histidine residues; circular dichroism revealed the occurrence of a substantial conformational rearrangement of Abeta(28); 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 Abeta(42). The implications of the above findings for a possible use of ruthenium compounds in Alzheimers disease are discussed.

Cooperative interactions between odorant-binding proteins of Anopheles gambiae

To understand olfactory discrimination in Anopheles gambiae, we made six purified recombinant OBPs and investigated their ligand-binding properties. All OBPs were expressed in bacteria with additional production of OBP47 in the yeast Kluveromyces lactis. Ligand-binding experiments, performed with a diverse set of organic compounds, revealed marked differences between the OBPs. Using the fluorescent probe N-phenyl-1-naphthylamine, we also measured the binding curves for binary mixtures of OBPs and obtained, in some cases, unexpected behaviour, which could only be explained by the OBPs forming heterodimers with binding characteristics different from those of the component proteins. This shows that OBPs in mosquitoes can form complexes with novel ligand specificities, thus amplifying the repertoire of OBPs and the number of semiochemicals that can be discriminated. Confirmation of the likely role of heterodimers was demonstrated by in situ hybridisation, suggesting that OBP1 and OBP4 are co-expressed in some antennal sensilla of A. gambiae.

Structural and solution chemistry, protein binding and antiproliferative profiles of gold(I)/(III) complexes bearing the saccharinato ligand

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 NH(4)(+)), [(PTA)Au(sac)], K[Au(sac)(3)Cl] 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)(3)Cl] 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.

Insights on the mechanism of thioredoxin reductase inhibition by gold N-heterocyclic carbene compounds using the synthetic linear selenocysteine containing C-terminal peptide hTrxR(488-499): an ESI-MS investigation.

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.

Cytotoxic Profile and Peculiar Reactivity with Biomolecules of a Novel "Rule-Breaker" Iodidoplatinum(II) Complex.

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.

Reactivity and biological properties of a series of cytotoxic PtI2(amine)2 complexes, either cis or trans configured.

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.

Cancer associated fibroblasts transfer lipids and proteins to cancer cells through cargo vesicles supporting tumor growth

Fibroblasts are the most abundant cells in connective tissue and, with fibrillar extracellular matrix, form the structural scaffolding of organs. In solid tumors, interaction with cancer cells induces fibroblasts transdifferentiation into an activated form, which become a fundamental part of the tumor stroma. Within tumor microenvironment stromal and cancer cells engage a crosstalk that is mediated by soluble factors, cellcell contacts and extracellular vesicles trafficlking. Here we report that fibroblasts have the ability to transfer a remarkable amount of proteins and lipids to neighboring cells, in an ectosome-dependent fashion, identifying a novel and native property of these cells. Cancer-associated fibroblasts show an enhanced production and delivering of ectc:Jsomes to cancer cells compared to normal fibroblasts. As a consequence of this phenomenon, tumor cells increase their proliferation rate, indicating that ectosome-mediated trafficking could be a relevant mechanism mediating the trophic function of activated connective tissue on tumor cells.