Non-Covalent and Covalent Binding of New Mixed-Valence Cage-like Polyoxidovanadate Clusters to Lysozyme.

The high-resolution X-ray structures of the model protein lysozyme in the presence of the potential drug [VIVO(acetylacetonato)2] from crystals grown in 1.1 M NaCl, 0.1 M sodium acetate at pH 4.0 reveal the binding to the protein of different and unexpected mixed-valence cage-like polyoxidovanadates (POVs): [V15O36(OH2)]5-, which non-covalently interacts with the lysozyme surface, [V15O33(OH2)]+ and [V20O51(OH2)]n- (this latter based on an unusual {V18O43} cage) which covalently bind the protein. EPR spectroscopy confirms the partial oxidation of VIV to VV and the formation of mixed-valence species. The results indicate that the interaction with proteins can stabilize the structure of unexpected - both for dimension and architecture - POVs, not observed in aqueous solution.

Protein-Protein Stabilization in VIVO/8-Hydroxyquinoline-Lysozyme adduct: A Spectroscopic, Crystallographic, and Computational Study.

The binding of the potential drug [VIVO(8-HQ)2], where 8-HQ is 8-hydroxyquinolinato, with hen egg white lysozyme (HEWL) was evaluated through spectroscopic (electron paramagnetic resonance, EPR, and UV-visible), spectrometric (electrospray ionization-mass spectrometry, ESI-MS), crystallographic (X-ray diffraction, XRD), and computational (DFT and docking) studies. ESI-MS indicates the interaction of [VIVO(8-HQ)(H2O)]+ and [VIVO(8-HQ)2(H2O)] species with HEWL. Room temperature EPR spectra suggest both covalent and non-covalent binding of the two different V-containing fragments. XRD analyses confirm these findings, showing that [VIVO(8-HQ)(H2O)]+ interacts covalently with the solvent exposed Asp119, while cis-[VIVO(8-HQ)2(H2O)] non-covalently with Arg128 and Lys96 from a symmetry mate. The covalent binding of [VIVO(8-HQ)(H2O)]+ to Asp119 is favored by a π-π contact with Trp62 and a H-bond with Asn103 of a symmetry-related molecule. Additionally, the covalent binding of VVO2+ to Asp48 and non-covalent binding of other V-containing fragments to Arg5, Cys6, and Glu7 is revealed. Molecular docking indicates that, in the absence of the interactions occurring at the protein-protein interface close to Asp119, the binding to Glu35 or Asp52 should be preferred. Such a protein-protein stabilization could be more common than what believed up today, at least in the solid state, and should be considered in the characterization of metal-protein adducts.

Picoplatin binding to proteins: X-ray structures and mass spectrometry data on the adducts with lysozyme and ribonuclease A.

The reactivity of the anticancer drug picoplatin (cis-amminedichlorido(2-methylpyridine)platinum(II) complex) with the model proteins hen egg white lysozyme (HEWL) and bovine pancreatic ribonuclease (RNase A) was investigated by electrospray ionisation mass spectrometry (ESI MS) and X-ray crystallography. The data were compared with those previously obtained for the adducts of these proteins with cisplatin, carboplatin and oxaliplatin under the same experimental conditions. ESI-MS data show binding of Pt to both proteins, with fragments retaining the 2-methylpyridine ligand and, possibly, a chloride ion. X-ray crystallography identifies different binding sites on the two proteins, highlighting a different behaviour of picoplatin in the absence or presence of dimethyl sulfoxide (DMSO). Metal-containing fragments bind to HEWL close to the side chains of His15, Asp18, Asp119 and both Lys1 and Glu7, whereas they bind to RNase A on the side chain of His12, Met29, His48, Asp53, Met79, His105 and His119. The data suggest that the presence of DMSO favours the loss of 2-methylpyridine and alters the ability of the Pt compound to bind to the two proteins. With both proteins, picoplatin appears to behave similarly to cisplatin and carboplatin when dissolved in DMSO, whereas it behaves more like oxaliplatin in the absence of the coordinating solvent. This study provides important insights into the pharmacological profile of picoplatin and supports the conclusion that coordinating solvents should not be used to evaluate the biological activities of Pt-based drugs.

Comparative Analysis of the Inhibitory Mechanism of Aß1-42 Aggregation by Diruthenium Complexes.

There is a growing interest in the search for metal-based therapeutics for protein misfolding disorders such as Alzheimer's disease (AD). A novel and largely unexplored class of metallodrugs is constituted by paddlewheel diruthenium complexes, which exhibit unusual water solubility and stability and unique coordination modes to proteins. Here, we investigate the ability of the complexes [Ru2Cl(DPhF)(O2CCH3)3]·H2O (1), [Ru2Cl(DPhF)2(O2CCH3)2]·H2O (2), and K2[Ru2(DPhF)(CO3)3]·3H2O (3) (DPhF- = N,N'-diphenylformamidinate) to interfere with the amyloid aggregation of the Aß1-42 peptide. These compounds differ in charge and steric hindrance due to the coordination of a different number of bulky ligands. The mechanisms of action of the three complexes were studied by employing a plethora of physicochemical and biophysical techniques as well as cellular assays. All these studies converge on different mechanisms of inhibition of amyloid fibrillation: complexes 1 and 2 show a clear inhibitory effect due to an exchange ligand process in the Ru2 unit aided by aromatic interactions. Complex 3 shows no inhibition of aggregation, probably due to its negative charge in solution. This study demonstrates that slight variations in the ligands surrounding the bimetallic core can modulate the amyloid aggregation inhibition and supports the use of paddlewheel diruthenium complexes as promising therapeutics for Alzheimer's disease.

Metal-based carbon monoxide releasing molecules with promising cytotoxic properties.

Carbon monoxide, the "silent killer" gas, is increasingly recognised as an important signalling molecule in human physiology, which has beneficial biological properties. A particular way of achieving controlled CO administration is based on the use of biocompatible molecules that only release CO when triggered by internal or external factors. These approaches include the development of pharmacologically effective prodrugs known as CO releasing molecules (CORMs), which can supply biological systems with CO in well-regulated doses. An overview of transition metal-based CORMs with cytotoxic properties is here reported. The mechanisms at the basis of the biological activities of these molecules and their potential therapeutical applications with respect to their stability and CO releasing properties have been discussed. The activation of metal-based CORMs is determined by the type of metal and by the nature and features of the auxiliary ligands, which affect the metal core electronic density and therefore the prodrug resistance towards oxidation and CO release ability. A major role in regulating the cytotoxic properties of these CORMs is played by CO and/or CO-depleted species. However, several mysteries concerning the cytotoxicity of CORMs remain as intriguing questions for scientists.

Structural insights and aggregation propensity of a super-stable monellin mutant: A new potential building block for protein-based nanostructured materials.

Protein fibrillation is commonly associated with pathologic amyloidosis. However, under appropriate conditions several proteins form fibrillar structures in vitro that can be used for biotechnological applications. MNEI and its variants, firstly designed as single chain derivatives of the sweet protein monellin, are also useful models for protein fibrillary aggregation studies. In this work, we have drawn attention to a protein dubbed Mut9, already characterized as a "super stable" MNEI variant. Comparative analysis of the respective X-ray structures revealed how the substitutions present in Mut9 eliminate several unfavorable interactions and stabilize the global structure. Molecular dynamic predictions confirmed the presence of a hydrogen-bonds network in Mut9 which increases its stability, especially at neutral pH. Thioflavin-T (ThT) binding assays and Fourier transform infrared (FTIR) spectroscopy indicated that the aggregation process occurs both at acidic and neutral pH, with and without addition of NaCl, even if with a different kinetics. Accordingly, Transmission Electron Microscopy (TEM) showed a fibrillar organization of the aggregates in all the tested conditions, albeit with some differences in the quantity and in the morphology of the fibrils. Our data underline the great potential of Mut9, which combines great stability in solution with the versatile conversion into nanostructured biomaterials.

On the mechanism of action of arsenoplatins: arsenoplatin-1 binding to a B-DNA dodecamer.

The reaction of Pt-based anticancer agents with arsenic trioxide affords robust complexes known as arsenoplatins. The prototype of this family of anticancer compounds is arsenoplatin-1 (AP-1) that contains an As(OH)2 fragment linked to a Pt(II) moiety derived from cisplatin. Crystallographic and spectrometric studies of AP-1 binding to a B-DNA double helix dodecamer are presented here, in comparison with cisplatin and transplatin. Results reveal that AP-1, cisplatin and transplatin react differently with the DNA model system. Notably, in the AP-1/DNA systems, the Pt-As bond can break down with time and As-containing fragments can be released. These results have implications for the understanding of the mechanism of action of arsenoplatins.

A Diruthenium Metallodrug as a Potent Inhibitor of Amyloid-ß Aggregation: Synergism of Mechanisms of Action.

The physical and chemical properties of paddlewheel diruthenium compounds are highly dependent on the nature of the ligands surrounding the bimetallic core. Herein, we compare the ability of two diruthenium compounds, [Ru2Cl(D-p-FPhF)(O2CCH3)3]·H2O (1) (D-p-FPhF- = N,N'-bis(4-fluorophenyl)formamidinate) and K3[Ru2(O2CO)4]·3H2O (2), to act as inhibitors of amyloid aggregation of the Aß1-42 peptide and its peculiar fragments, Aß1-16 and Aß21-40. A wide range of biophysical techniques has been used to determine the inhibition capacity against aggregation and the possible mechanism of action of these compounds (Thioflavin T fluorescence and autofluorescence assays, UV-vis absorption spectroscopy, circular dichroism, nuclear magnetic resonance, mass spectrometry, and electron scanning microscopy). Data show that the most effective inhibitory effect is shown for compound 1. This compound inhibits fiber formation and completely abolishes the cytotoxicity of Aß1-42. The antiaggregatory capacity of this complex can be explained by a binding mechanism of the dimetallic units to the peptide chain along with π-π interactions between the formamidinate ligand and the aromatic side chains. The results suggest the potential use of paddlewheel diruthenium complexes as neurodrugs and confirm the importance of the steric and charge effects on the properties of diruthenium compounds.

Stabilization and Binding of [V4 O12 ]4- and Unprecedented [V20 O54 (NO3 )]n- to Lysozyme upon Loss of Ligands and Oxidation of the Potential Drug VIV O(acetylacetonato)2.

High-resolution crystal structures of lysozyme in the presence of the potential drug VIV O(acetylacetonato)2 under two different experimental conditions have been solved. The crystallographic study reveals the loss of the ligands, the oxidation of VIV to VV and the subsequent formation of adducts of the protein with two different polyoxidovanadates: [V4 O12 ]4- , which interacts with lysozyme non-covalently, and the unprecedented [V20 O54 (NO3 )]n- , which is covalenty bound to the side chain of an aspartate residue of symmetry related molecules.

Steric hindrance and charge influence on the cytotoxic activity and protein binding properties of diruthenium complexes.

Paddlewheel diruthenium complexes are being used as metal-based drugs. It has been proposed that their charge and steric properties determine their selectivity towards proteins. Here, we explore these parameters using the first water-soluble diruthenium complex bearing two formamidinate ligands, [Ru2Cl(DPhF)2(O2CCH3)2], and two derivatives, [Ru2Cl(DPhF)(O2CCH3)3] and K2[Ru2(DPhF)(CO3)3] (DPhF- = N,N'-diphenylformamidinate), with one formamidinate. Their protein binding properties have been assessed employing hen egg white lysozyme (HEWL). The results confirm the relationship between the type of interaction (coordinate/non-coordinate bonds) and the charge of diruthenium complexes. The crystallization medium is also a key factor. In all cases, diruthenium species maintain the M-M bond and produce stable adducts. The antiproliferative properties of these diruthenium complexes have been evaluated on an eukaryotic cell-based model. Our data show a correlation between the number of the formamidinate ligands and the anticancer activity of the diruthenium derivatives against human epithelial carcinoma cells. Increased cytotoxicity may be related to increased steric hindrance and Ru25+ core electronic density. However, the effect of increasing the lipophilicity of diruthenium species by introducing a second N,N'-diphenylformamidinate must be also considered. This work illustrates a systematic approach to shed light on the relevant properties of diruthenium compounds to design metal-based metallodrugs and diruthenium metalloenzymes.

Metal-Complexes Bearing Releasable CO Differently Modulate Amyloid Aggregation.

Neurodegenerative diseases are often associated with an uncontrolled amyloid aggregation. Hence, many studies are oriented to discover new compounds that are able to modulate self-recognition mechanisms of proteins involved in the development of these pathologies. Herein, three metal-complexes able to release carbon monoxide (CORMs) were analyzed for their ability to affect the self-aggregation of the amyloidogenic fragment of nucleophosmin 1, corresponding to the second helix of the three-helix bundle located in the C-terminal domain of the protein, i.e., NPM1264-277, peptide. These complexes were two cymantrenes coordinated to the nucleobase adenine (Cym-Ade) and to the antibiotic ciprofloxacin (Cym-Cipro) and a Re(I)-compound containing 1,10-phenanthroline and 3-CCCH2NHCOCH2CH2-6-bromo-chromone as ligands (Re-Flavo). Thioflavin T (ThT) assay, UV-vis absorption and fluorescence spectroscopies, scanning electron microscopy (SEM), and electrospray ionization mass spectrometry (ESI-MS) indicated that the three compounds have different effects on the peptide aggregation. Cym-Ade and Cym-Cipro act as aggregating agents. Cym-Ade induces the formation of NPM1264-277 fibers longer and stiffer than that formed by NPM1264-277 alone; irradiation of complexes speeds the formation of fibers that are more flexible and thicker than those found without irradiation. Cym-Cipro induces the formation of longer fibers, although slightly thinner in diameter. Conversely, Re-Flavo acts as an antiaggregating agent. Overall, these results indicate that metal-based CORMs with diverse structural features can have a different effect on the formation of amyloid fibers. A proper choice of ligands attached to metal can allow the development of metal-based drugs with potential application as antiamyloidogenic agents.

Cisplatin binding to angiogenin protein: new molecular pathways and targets for the drug's anticancer activity.

Cisplatin (CisPt), a platinum-based chemotherapeutic widely used in the treatment of various cancers, has multiple mechanisms of action, including nuclear DNA (nDNA) and mitochondrial DNA (mDNA) damage and cytoskeletal perturbations affecting, in turn, the membrane transporter activity. CisPt binding to proteins and enzymes may modulate its biochemical mechanism of action and is associated with cancer cell resistance to the drug. In this work, we investigate the interaction between cisplatin and angiogenin (Ang), a protein strongly expressed in many types of cancer and a potent angiogenic factor. The adduct formed upon reaction of CisPt with Ang (Ang@CisPt) was characterized by X-ray crystallography to evidence the exact platination site and by UV-visible (UV-vis) absorption and circular dichroism (CD) spectroscopies to shed light on any possible change in the protein conformation. Furthermore, high-resolution electrospray ionization (ESI) mass spectrometry was utilized to evaluate the Ang : CisPt stoichiometry of the Ang@CisPt adduct. The effect of the Ang@CisPt adduct on a prostate cancer cell line (PC-3) was tested by colorimetric assays in terms of cell viability, at both levels of nuclear and mitochondrial damage, and reactive oxygen species (ROS) production. Cellular imaging by laser scanning confocal microscopy (LSM) was utilized to scrutinize the cytoskeleton actin reorganization and the lysosome and mitochondria organelle perturbation. These studies highlight the possibility of new molecular pathways and targets for CisPt activity.

Implications of Protein Interaction in the Speciation of Potential VIVO-Pyridinone Drugs.

Vanadium complexes (VCs) are promising agents for the treatment, among others, of diabetes and cancer. The development of vanadium-based drugs is mainly limited by a scarce knowledge of the active species in the target organs, which is often determined by the interaction of VCs with biological macromolecules like proteins. Here, we have studied the binding of [VIVO(empp)2] (where Hempp is 1-methyl-2-ethyl-3-hydroxy-4(1H)-pyridinone), an antidiabetic and anticancer VC, with the model protein hen egg white lysozyme (HEWL) by electrospray ionization-mass spectrometry (ESI-MS), electron paramagnetic resonance (EPR), and X-ray crystallography. ESI-MS and EPR techniques reveal that, in aqueous solution, both the species [VIVO(empp)2] and [VIVO(empp)(H2O)]+, derived from the first one upon the loss of a empp(-) ligand, interact with HEWL. Crystallographic data, collected under different experimental conditions, show covalent binding of [VIVO(empp)(H2O)]+ to the side chain of Asp48, and noncovalent binding of cis-[VIVO(empp)2(H2O)], [VIVO(empp)(H2O)]+, [VIVO(empp)(H2O)2]+, and of an unusual trinuclear oxidovanadium(V) complex, [VV3O6(empp)3(H2O)], with accessible sites on the protein surface. The possibility of covalent and noncovalent binding with different strength and of interaction with various sites favor the formation of adducts with the multiple binding of vanadium moieties, allowing the transport in blood and cellular fluids of more than one metal-containing species with a possible amplification of the biological effects.

Dirhodium tetraacetate binding to a B-DNA double helical dodecamer probed by X-ray crystallography and mass spectrometry.

The reaction of the cytotoxic compound dirhodium tetraacetate with a B-DNA double helical dodecamer was studied by X-ray crystallography and mass spectrometry. The structure of the dirhodium/DNA adduct reveals a dimetallic center binding to an adenine via axial coordination. Complementary information has been gained through ESI MS measurements. Comparison between the present data and those previously obtained for cisplatin indicates that the two metallodrugs react with this DNA dodecamer in a significantly different fashion.

Cross-Linked Crystals of Dirhodium Tetraacetate/RNase A Adduct Can Be Used as Heterogeneous Catalysts.

Due to their unique coordination structure, dirhodium paddlewheel complexes are of interest in several research fields, like medicinal chemistry, catalysis, etc. Previously, these complexes were conjugated to proteins and peptides for developing artificial metalloenzymes as homogeneous catalysts. Fixation of dirhodium complexes into protein crystals is interesting to develop heterogeneous catalysts. Porous solvent channels present in protein crystals can benefit the activity by increasing the probability of substrate collisions at the catalytic Rh binding sites. Toward this goal, the present work describes the use of bovine pancreatic ribonuclease (RNase A) crystals with a pore size of 4 nm (P3221 space group) for fixing [Rh2(OAc)4] and developing a heterogeneous catalyst to perform reactions in an aqueous medium. The structure of the [Rh2(OAc)4]/RNase A adduct was investigated by X-ray crystallography: the metal complex structure remains unperturbed upon protein binding. Using a number of crystal structures, metal complex accumulation over time, within the RNase A crystals, and structures at variable temperatures were evaluated. We also report the large-scale preparation of microcrystals (∼10-20 µm) of the [Rh2(OAc)4]/RNase A adduct and cross-linking reaction with glutaraldehyde. The catalytic olefin cyclopropanation reaction and self-coupling of diazo compounds by these cross-linked [Rh2(OAc)4]/RNase A crystals were demonstrated. The results of this work reveal that these systems can be used as heterogeneous catalysts to promote reactions in aqueous solution. Overall, our findings demonstrate that the dirhodium paddlewheel complexes can be fixed in porous biomolecule crystals, like those of RNase A, to prepare biohybrid materials for catalytic applications.

Impact of Hydrophobic Chains in Five-Coordinate Glucoconjugate Pt(II) Anticancer Agents.

This study describes new platinum(II) cationic five-coordinate complexes (1-R,R') of the formula [PtR(NHC)(dmphen)(ethene)]CF3SO3 (dmphen = 2,9-dimethyl-1,10-phenanthroline), containing in their axial positions an alkyl group R (methyl or octyl) and an imidazole-based NHC-carbene ligand with a substituent R' of variable length (methyl or octyl) on one nitrogen atom. The Pt-carbene bond is stable both in DMSO and in aqueous solvents. In DMSO, a gradual substitution of dmphen and ethene is observed, with the formation of a square planar solvated species. Octanol/water partitioning studies have revealed the order of hydrophobicity of the complexes (1-Oct,Me > 1-Oct,Oct > 1-Me,Oct > 1-Me,Me). Their biological activity was investigated against two pairs of cancer and non-cancer cell lines. The tested drugs were internalized in cancer cells and able to activate the apoptotic pathway. The reactivity of 1-Me,Me with DNA and protein model systems was also studied using UV-vis absorption spectroscopy, fluorescence, and X-ray crystallography. The compound binds DNA and interacts in various ways with the model protein lysozyme. Remarkably, structural data revealed that the complex can bind lysozyme via non-covalent interactions, retaining its five-coordinate geometry.

Inside out Porphyridium cruentum: Beyond the Conventional Biorefinery Concept.

Here, an unprecedented biorefinery approach has been designed to recover high-added value bioproducts starting from the culture ofPorphyridium cruentum. This unicellular marine red alga can secrete and accumulate high-value compounds that can find applications in a wide variety of industrial fields. 300 ± 67 mg/L of exopolysaccharides were obtained from cell culture medium; phycoerythrin was efficiently extracted (40% of total extract) and isolated by single chromatography, with a purity grade that allowed the crystal structure determination at 1.60 Å; a twofold increase in ß-carotene yield was obtained from the residual biomass; the final residual biomass was found to be enriched in saturated fatty acids. Thus, for the first time, a complete exploitation ofP. cruentumculture was set up.

Cisplatin Binding to Human Serum Transferrin: A Crystallographic Study.

The molecular mechanism of how human serum transferrin (hTF) recognizes cisplatin at the atomic level is still unclear. Here, we report the molecular structure of the adduct formed upon the reaction of hTF with cisplatin. Pt binds the side chain of Met256 (at the N-lobe), without altering the protein overall conformation.

Effect of Equatorial Ligand Substitution on the Reactivity with Proteins of Paddlewheel Diruthenium Complexes: Structural Studies.

The paddlewheel [Ru2Cl(O2CCH3)4] complex was previously reported to react with the model protein hen egg white lysozyme (HEWL), forming adducts with two diruthenium moieties bound to Asp101 and Asp119 side chains upon the release of one acetate. To study the effect of the equatorial ligands on the reactivity with proteins of diruthenium compounds, X-ray structures of the adducts formed when HEWL reacts with [Ru2Cl(D-p-FPhF)(O2CCH3)3] [D-p-FPhF = N,N'-bis(4-fluorophenyl)formamidinate] under different conditions were solved. [Ru2Cl(D-p-FPhF)(O2CCH3)3] is bonded through their equatorial positions to the Asp side chains. Protein binding occurs cis or trans to D-p-FPhF. Lys or Arg side chains or even main-chain carbonyl groups can coordinate to the diruthenium core at the axial site. Data help to understand the reactivity of paddlewheel diruthenium complexes with proteins, providing useful information for the design of new artificial diruthenium-containing metalloenzymes with potential applications in the fields of catalysis, biomedicine, and biotechnology.

A new and efficient procedure to load bioactive molecules within the human heavy-chain ferritin nanocage.

For their easy and high-yield recombinant production, their high stability in a wide range of physico-chemical conditions and their characteristic hollow structure, ferritins (Fts) are considered useful scaffolds to encapsulate bioactive molecules. Notably, for the absence of immunogenicity and the selective interaction with tumor cells, the nanocages constituted by the heavy chain of the human variant of ferritin (hHFt) are optimal candidates for the delivery of anti-cancer drugs. hHFt nanocages can be disassembled and reassembled in vitro to allow the loading of cargo molecules, however the currently available protocols present some relevant drawbacks. Indeed, protein disassembly is achieved by exposure to extreme pH (either acidic or alkaline), followed by incubation at neutral pH to allow reassembly, but the final protein recovery and homogeneity are not satisfactory. Moreover, the exposure to extreme pH may affect the structure of the molecule to be loaded. In this paper, we report an alternative, efficient and reproducible procedure to reversibly disassemble hHFt under mild pH conditions. We demonstrate that a small amount of sodium dodecyl sulfate (SDS) is sufficient to disassemble the nanocage, which quantitatively reassembles upon SDS removal. Electron microscopy and X-ray crystallography show that the reassembled protein is identical to the untreated one. The newly developed procedure was used to encapsulate two small molecules. When compared to the existing disassembly/reassembly procedures, our approach can be applied in a wide range of pH values and temperatures, is compatible with a larger number of cargos and allows a higher protein recovery.