JBIC Journal of Biological Inorganic Chemistry (v.23, #8)

Potential applications of engineered nanoparticles in medicine and biology: an update by Gudepalya Renukaiah Rudramurthy; Mallappa Kumara Swamy (1185-1204).
Nanotechnology advancements have led to the development of its allied fields, such as nanoparticle synthesis and their applications in the field of biomedicine. Nanotechnology driven innovations have given a hope to the patients as well as physicians in solving the complex medical problems. Nanoparticles with a size ranging from 0.2 to 100 nm are associated with an increased surface to volume ratio. Moreover, the physico-chemical and biological properties of nanoparticles can be modified depending on the applications. Different nanoparticles have been documented with a wide range of applications in various fields of medicine and biology including cancer therapy, drug delivery, tissue engineering, regenerative medicine, biomolecules detection, and also as antimicrobial agents. However, the development of stable and effective nanoparticles requires a profound knowledge on both physico-chemical features of nanomaterials and their intended applications. Further, the health risks associated with the use of engineered nanoparticles needs a serious attention.
Keywords: Biomedicine; Biomolecule detection; Cancer therapy; Drug delivery; Engineered nanoparticles; Regenerative medicine; Tissue engineering

Polynuclear ruthenium organometallic complexes containing a 1,3,5-triazine ligand: synthesis, DNA interaction, and biological activity by Floyd A. Beckford; Madison B. Niece; Brittany P. Lassiter; Stephen J. Beebe; Alvin A. Holder (1205-1217).
It is now well established that ruthenium complexes are attractive alternatives to platinum-based anticancer agents. Most of the ruthenium compounds currently under investigation contain a single metal center. The synthesis of multinuclear analogues may provide access to novel complexes with enhanced biological activity. In this work, we have synthesized a set of three trinuclear complexes containing organometallic ruthenium fragments—(arene)RuCl—coordinated to a 2,4,6-tris(di-2-pyridylamino)-1,3,5-triazine core [(Arene = benzene (2), p-cymene (1), or hexamethylbenzene (3)]. The interaction of the complexes with DNA was extensively studied using a variety of biophysical probes as well as by molecular docking. The complexes bind strongly to DNA with apparent binding constants ranging from 2.20 to 4.79 × 104 M−1. The binding constants from electronic absorption titrations were an order of magnitude greater. The mode of binding to the nucleic acid was not definitively determined, but the evidence pointed to some kind of non-specific electrostatic interaction. None of the complexes displayed any significant antimicrobial activity against the organisms that were studied and exhibited anticancer activity only at high (> 100 μM) concentration.
Keywords: Ruthenium; Organometallic; DNA; Molecular docking; Multinuclear

Recent evidence links the role of human glutaminyl cyclase (hQC) to the amyloidogenic process involved in Alzheimer’s disease (AD). hQC is a zinc enzyme present in neuronal tissue and its activity is responsible for the cyclization of N-terminal Gln or Glu β-amyloid peptides, leading to N-pyroglutamic acid peptides (pE-Aβ) that is probably a crucial event in the initiation and progress of the disease. Indeed, pE-containing peptides exhibit an elevated neurotoxicity and a tendency to aggregate. These observations render hQC inhibition an attractive strategy for developing new molecules active against AD. We present here the crystal structure of hQC in complex with SEN177, a newly designed molecule. The SEN177-binding mode to hQC differs from that of the known hQC inhibitors. SEN177 K i on hQC is 20 nM, comparable or better than that of the most potent known hQC inhibitors PBD150 and PQ912. In addition, SEN177 already demonstrated relevant pharmacological properties in in vivo models of Huntington’s disease. All these properties make SEN177 an important scaffold for developing molecules acting on AD and related diseases.
Keywords: Alzheimer’s disease; Pyroglutamate; Glutaminyl cyclase; Inhibitor; X-ray crystallography

Aroylhydrazones constitute a promising class of ‘metal-protein attenuating compounds’ for the treatment of Alzheimer’s disease: a proof-of-concept based on the study of the interactions between zinc(II) and pyridine-2-carboxaldehyde isonicotinoyl hydrazone by Daphne S. Cukierman; Elio Accardo; Rosana Garrido Gomes; Anna De Falco; Marco C. Miotto; Maria Clara Ramalho Freitas; Mauricio Lanznaster; Claudio O. Fernández; Nicolás A. Rey (1227-1241).
With the increasing life expectancy of the world’s population, neurodegenerative diseases, such as Alzheimer’s disease (AD), will become a much more relevant public health issue. This fact, coupled with the lack of efficacy of the available treatments, has been driving research directed to the development of new drugs for this pathology. Metal-protein attenuating compounds (MPACs) constitute a promising class of agents with potential application on the treatment of neurodegenerative diseases, such as AD. Currently, most MPACs are based on 8-hydroxyquinoline. Recently, our research group has described the hybrid aroylhydrazone containing the 8-hydroxyquinoline group INHHQ as a promising MPAC. By studying the known structure-related ligand HPCIH, which does not contain the phenol moiety, as a simplified chemical model for INHHQ, we aimed to clarify the real impact of the aroylhydrazone group for the MPAC activity of a compound with potential anti-Alzheimer’s activity. The present work describes a detailed solution and solid-state study of the coordination of HPCIH with Zn2+ ions, as well as its in vitro binding-ability towards this metal in the presence of the Aβ(1–40) peptide. Similar to INHHQ, HPCIH is able to efficiently compete with Aβ(1–40) for Zn2+ ions, performing as expected for an MPAC. The similarity between the behaviors of both ligands is remarkable. Taken together, the data presented herein point to aroylhydrazones, such as the compounds HPCIH and the previously published INHHQ, as encouraging MPACs for the treatment of AD.
Keywords: Aroylhydrazones; MPAC; Alzheimer’s disease; Zinc(II); Aβ peptide

Formate dehydrogenases (FDHs) are metalloenzymes that catalyse the reversible conversion of formate to carbon dioxide. Since such a process may be used to combat the greenhouse effect, FDHs have been extensively studied by experimental and theoretical methods. However, the reaction mechanism is still not clear; instead five putative mechanisms have been suggested. In this work, the reaction mechanism of FDH was studied by computational methods. Combined quantum mechanical and molecular mechanic (QM/MM) optimisations were performed to obtain the geometries. To get more accurate energies and obtain a detailed account of the surroundings, big-QM calculations with a very large (1121 atoms) QM region were performed. Our results indicate that the formate substrate does not coordinate directly to Mo when it enters the oxidised active site of the FDH, but instead resides in the second coordination sphere. The sulfido ligand abstracts a hydride ion from the substrate, giving a Mo(IV)–SH state and a thiocarbonate ion attached to Cys196. The latter releases CO2 when the active site is oxidised back to the resting (MoVI) state. This mechanism is supported by recent experimental studies.
Keywords: Formate dehydrogenase; Density functional theory; Sulfur-shift mechanism; Molybdenum; QM/MM

Zn2+-binding in the glutamate-rich region of the intrinsically disordered protein prothymosin-alpha by Sriramya Garapati; William Monteith; Chris Wilson; Anastasiia Kostenko; John M. Kenney; Allison S. Danell; Colin S. Burns (1255-1263).
Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein’s function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein’s ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise K d values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.
Keywords: Prothymosin-alpha; Zinc binding; Intrinsically disordered protein; Circular dichroism; Mass spectrometry

New heteroleptic oxidovanadium(V) complexes: synthesis, characterization and biological evaluation as potential agents against Trypanosoma cruzi by Gonzalo Scalese; Ignacio Machado; Carolina Fontana; Gastón Risi; Gustavo Salinas; Leticia Pérez-Díaz; Dinorah Gambino (1265-1281).
Searching for prospective vanadium-based agents against Trypanosoma cruzi, the parasite causing Chagas disease, four new [VVO(8HQ–H)(L–2H)] compounds, where 8HQ is 8-hydroxyquinoline and L are tridentate salicylaldehyde semicarbazone derivatives L1–L4, were synthesized and characterized in the solid state and in solution. The compounds were evaluated on T. cruzi epimastigotes (CL Brener) as well as on VERO cells, as mammalian cell model. Compounds showed activity against T. cruzi (IC50 6.2–10.5 μM) of the same order than Nifurtimox and 8HQ, and a four- to sevenfold activity increase with respect to the free semicarbazones. For comparison, [VVO2(L–H)] series was prepared and the new [VVO2(L3–H)] was fully characterized. They showed negligible activity and low selectivity towards the parasite. The inclusion of 8HQ as ligand in [VVO(8HQ–H)(L–2H)] compounds led to good activities and increased selectivity towards the parasite with respect to 8HQ. 51V NMR experiments, performed to get insight into the nature of the active species, suggested partial decomposition of the compounds in solution to [VVO2(L–H)] and 8HQ. Depending on the dose, the compounds act as trypanocide or trypanostatic. A high uptake of vanadium in the parasites (58.51–88.9% depending on dose) and a preferential accumulation in the soluble protein fraction of the parasite was determined. Treated parasites do not seem to show a late apoptotic/necrotic phenotype suggesting a different cell death mechanism. In vivo toxicity study on zebrafish model showed no toxicity up to a 25 µM concentration of [VVO(8HQ–H)(L1–2H)]. These compounds could be considered prospective anti-T. cruzi agents that deserve further research.
Keywords: Vanadium; 8-Hydroxyquinoline; Semicarbazones; Trypanosoma cruzi ; Metallomics

Gold complexes are promising compounds used in cancer chemotherapy. Besides their steric features, which enable biomolecule interactions, the redox instability and the high affinity of gold with cellular nucleophiles influence the biological action in these complexes. Both features were herein theoretically investigated for the [Au(C^N^C)Cl] probe complex (C^N^C = 2,6-diphenylpyridine) using H2O, CH3SH/CH3S, CH3Se and meim-4-H (4-methylimidazole) as biomimetic nucleophiles. Based on the results, the lowest energy reaction path followed two consecutive steps: (1) chloride-exchange ($$k_{I}$$ kI  = 4.14 × 107 M−1 s−1) and (2) reduction of the resulting Au(III) metabolite to the corresponding Au(I) analog with chelate ring-opening ($$varepsilon_{3}^{o}$$ ε3o  =+0.15 V—data based on the reaction with CH3Se). These findings bring new insights about the mechanism of the Au(III) complex/biomolecule interaction in the cell, which is responsible for triggering biological responses.
Keywords: Au(III)-complexes; Activation-reaction; Reduction potential; DFT

A novel thermophilic hemoprotein scaffold for rational design of biocatalysts by Joana Efua Aggrey-Fynn; Nur Basak Surmeli (1295-1307).
Hemoproteins are commonly found in nature, and involved in many important cellular processes such as oxygen transport, electron transfer, and catalysis. Rational design of hemoproteins can not only inspire novel biocatalysts but will also lead to a better understanding of structure–function relationships in native hemoproteins. Here, the heme nitric oxide/oxygen-binding protein from Caldanaerobacter subterraneus subsp. tengcongensis (TtH-NOX) is used as a novel scaffold for oxidation biocatalyst design. We show that signaling protein TtH-NOX can be reengineered to catalyze H2O2 decomposition and oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) by H2O2. In addition, the role of the distal tyrosine (Tyr140) in catalysis is investigated. The mutation of Tyr140 to alanine hinders the catalysis of the oxidation reactions. On the other hand, the mutation of Tyr140 to histidine, which is commonly observed in peroxidases, leads to a significant increase of the catalytic activity. Taken together, these results show that, while the distal histidine plays an important role in hemoprotein reactions with H2O2, it is not always essential for oxidation activity. We show that TtH-NOX protein can be used as an alternative scaffold for the design of novel biocatalysts with desired reactivity or functionality.H-NOX proteins are homologous to the nitric oxide sensor soluble guanylate cyclase. Here, we show that the gas sensor protein TtH-NOX shows limited capacity for catalysis of redox reactions and it can be used as a novel scaffold in biocatalysis design.
Keywords: Hemoproteins; Protein engineering; Heme nitric oxide/oxygen-binding protein; Peroxidase; Enzymes

Structure and dynamics of Helicobacter pylori nickel-chaperone HypA: an integrated approach using NMR spectroscopy, functional assays and computational tools by Chris A. E. M. Spronk; Szymon Żerko; Michał Górka; Wiktor Koźmiński; Benjamin Bardiaux; Barbara Zambelli; Francesco Musiani; Mario Piccioli; Priyanka Basak; Faith C. Blum; Ryan C. Johnson; Heidi Hu; D. Scott Merrell; Michael Maroney; Stefano Ciurli (1309-1330).
Helicobacter pylori HypA (HpHypA) is a metallochaperone necessary for maturation of [Ni,Fe]-hydrogenase and urease, the enzymes required for colonization and survival of H. pylori in the gastric mucosa. HpHypA contains a structural Zn(II) site and a unique Ni(II) binding site at the N-terminus. X-ray absorption spectra suggested that the Zn(II) coordination depends on pH and on the presence of Ni(II). This study was performed to investigate the structural properties of HpHypA as a function of pH and Ni(II) binding, using NMR spectroscopy combined with DFT and molecular dynamics calculations. The solution structure of apo,Zn-HpHypA, containing Zn(II) but devoid of Ni(II), was determined using 2D, 3D and 4D NMR spectroscopy. The structure suggests that a Ni-binding and a Zn-binding domain, joined through a short linker, could undergo mutual reorientation. This flexibility has no physiological effect on acid viability or urease maturation in H. pylori. Atomistic molecular dynamics simulations suggest that Ni(II) binding is important for the conformational stability of the N-terminal helix. NMR chemical shift perturbation analysis indicates that no structural changes occur in the Zn-binding domain upon addition of Ni(II) in the pH 6.3–7.2 range. The structure of the Ni(II) binding site was probed using 1H NMR spectroscopy experiments tailored to reveal hyperfine-shifted signals around the paramagnetic metal ion. On this basis, two possible models were derived using quantum-mechanical DFT calculations. The results provide a comprehensive picture of the Ni(II) mode to HpHypA, important to rationalize, at the molecular level, the functional interactions of this chaperone with its protein partners.
Keywords: Metallochaperones; Metal transport; Molecular dynamics; Nuclear magnetic resonance; Computational chemistry; Nickel

DNA binding, cleavage and cytotoxicity studies of three mononuclear Cu(II) chloro-complexes containing N–S donor Schiff base ligands by Sidhali U. Parsekar; Joseph Fernandes; Arnab Banerjee; Om Prakash Chouhan; Sumit Biswas; Manohar Singh; Durga P. Mishra; Manjuri Kumar (1331-1349).
We report the biological activity of three Cu(II) complexes [Cu(pabt)Cl] (1), [Cu(pma)Cl] (2), and [Cu(pdta)Cl]Cl (3) (pabt = N-(2-mercaptophenyl)-2′-pyridylmethylenimine, pma = N-(2-pyridylmethyl)-2-mercaptoaniline, pdta = 2,2′-di(pyridyl-2-methyleneimine)diphenyl disulfide). 13 display four-line EPR multiplet in solution at RT suggesting that these are mononuclear. DNA-binding studies using spectrophotometric titration of these complexes with calf thymus DNA showed binding through intercalation mode which was found to be consistent with the observation of increased viscosity of DNA and quenching of fluorescence of ethidium bromide bound DNA in the presence of these complexes. All three complexes were found to be efficient in bringing about oxidative and hydrolytic cleavage of DNA. The proposed mechanism of hydrolytic DNA cleavage has been discussed. MTT assay showed remarkable cytotoxicity on cervical cancer HeLa cell line and the IC50 values were 1.27, 4.13, and 3.92 μM for 1, 2 and 3, respectively, as compared to the IC50 value (13 μM) reported for cisplatin in HeLa cells. AO/PI and Annexin-V/PI assay suggest the induction of cell death primarily via apoptotic pathway. Nuclear staining using DAPI was used to assess changes in nuclear morphology during apoptotic cell death. The role of reactive oxygen species (ROS) for induction of apoptotic cell death was studied using H2DCF-DA assay and the result suggests that the generation of ROS by the complexes may be a possible cause for their antiproliferative activity. TUNEL assay showed DNA fragmentation in apoptotic cells. Cell cycle analysis using flow cytometry showed significant increase in the G2/M phase in HeLa cells by the compounds 13.Mononuclear Cu(II) complexes display remarkable cytotoxicity against cervical cancer HeLa cell line. The generation of ROS by the complexes may be a cause of their antiproliferative activity. Fluorescent images from DAPI staining assay revealed that the cells undergoing apoptosis displayed typical features like cell shrinkage, membrane blebbing, chromatin condensation and nuclear fragmentation. TUNEL assay showed DNA fragmentation in apoptotic cells.
Keywords: Cu(II) complexes; DNA binding and cleavage; MTT assay; TUNEL assay; Cytotoxicity; Anticancer activity