Carnoquinolines Target Copper Dyshomeostasis, Aberrant Protein-Protein Interactions, and Oxidative Stress.

Metal dysregulation, oxidative stress, protein modification, and aggregation are factors strictly interrelated and associated with neurodegenerative pathologies. As such, all of these aspects represent valid targets to counteract neurodegeneration and, therefore, the development of metal-binding compounds with other properties to combat multifactorial disorders is definitely on the rise. Herein, the synthesis and in-depth analysis of the first hybrids of carnosine and 8-hydroxyquinoline, carnoquinolines (CarHQs), which combine the properties of the dipeptide with those of 8-hydroxyquinoline, are reported. CarHQs and their copper complexes were characterized through several techniques, such as ESI-MS and NMR, UV/Vis, and circular dichroism spectroscopy. CarHQs can modulate self- and copper-induced amyloid-ß aggregation. These hybrids combine the antioxidant activity of their parent compounds. Therefore, they can simultaneously scavenge free radicals and reactive carbonyl species, thanks to the phenolic group and imidazole ring. These results indicate that CarHQs are promising multifunctional candidates for neurodegenerative disorders and they are worthy of further studies.

Complexation of As(III) by phosphonate ligands in aqueous fluids: Thermodynamic behavior, chemical binding forms and sequestering abilities.

In recent years, the contamination of water by arsenic reached alarming levels in many countries of the world, attracting the interest of many researchers engaged in testing methodologies able to remove this harmful pollutant. An important aspect that must be taken into consideration is the possibility to find arsenic in different chemical forms which could require different approaches for its removal. At this aim, a speciation analysis appears to be crucial for better understanding the behavior of arsenic species in aqueous solutions, especially in presence of compounds with marked chelating properties. Phosphonates can be identified as good sequestering agents and, at this purpose, this manuscript intends to investigate the interaction of As(III) with three phosphonic acids derived from nitrilotriacetic acid (NTA) by replacements of one (N-(Phosphonomethyl) iminodiacetic acid, NTAP), two (N,N-Bis-(phosphonomethyl) glycine, NTA2P) and three (Nitrilotri(methylphosphonic acid), NTA3P) carboxylic groups with the same number of phosphonate groups. An in-depth potentiometric and calorimetric investigation allowed to determine speciation models featured by simple ML, MLHi and ML(OH) species. A complete thermodynamic characterization of the systems is reported together with the definition of coordination mode by mass spectrometry measurements. On the light of the speciation models, the possibility of using these ligands in arsenic removal techniques was assessed by determining the pL0.5 (the concentration of ligand able to remove the 50% of metal ion present in trace). All ligands show a good sequestering ability, in particular under the conditions of fresh water, following the trend NTA3P > NTA2P > NTAP.

Zinc(II) complexes of ubiquitin: speciation, affinity and binding features.

Intraneuronal inclusions consisting of hypermetallated, (poly-)ubiquitinated proteins are a hallmark of neurodegeneration. To highlight the possible role played by metal ions in the dysfunction of the ubiquitin-proteasome system, here we report on zinc(II)/ubiquitin binding in terms of affinity constants, speciation, preferential binding sites and effects on protein stability and self-assembly. Potentiometric titrations allowed us to establish that at neutral pH only two species, ZnUb and Zn(2)Ub, are present in solution, in line with ESI-MS data. A change in the diffusion coefficient of ubiquitin was observed by NMR DOSY experiments after addition of Zn(II) ions, and thus indicates metal-promoted formation of protein assemblies. Analysis of (1)H, (15)N, (13)Cα and (13)CO chemical-shift perturbation after equimolar addition of Zn(II) ions to ubiquitin outlined two different metal-binding modes. The first involves a dynamic equilibrium in which zinc(II) is shared between a region including Met1, Gln2, Ile3, Phe4, Thr12, Leu15, Glu16, Val17, Glu18, Ile61 and Gln62 residues, which represent a site already described for copper binding, and a domain comprising Ile23, Glu24, Lys27, Ala28, Gln49, Glu51, Asp52, Arg54 and Thr55 residues. A second looser binding mode is centred on His68. Differential scanning calorimetry evidenced that addition of increasing amounts of Zn(II) ions does not affect protein thermal stability; rather it influences the shape of thermograms because of the increased propensity of ubiquitin to self-associate. The results presented here indicate that Zn(II) ions may interact with specific regions of ubiquitin and promote protein-protein contacts.

Noncovalent interaction-driven stereoselectivity of copper(II) complexes with cyclodextrin derivatives of L- and D-carnosine.

L-Carnosine (ß-alanyl-L-histidine, LCar) is the most widely and abundantly distributed copper(II)-coordinating endogenous dipeptide. Though its physiological role has not been completely understood yet, many functions have been proposed for this compound. LCar might be crucial in the potential reduction or prevention of several pathologies in which the metal ions are thought to be involved. The potential therapeutic applications of LCar are drastically limited because of hydrolysis by specific dipeptidases (carnosinases). D-Carnosine (DCar), the enantiomer of the naturally occurring dipeptide, shows the same properties as those of LCar, but it is not hydrolyzed by carnosinases. Chemical modification of LCar has been proposed as a promising strategy to reduce its enzymatic hydrolysis; conjugation of a carbohydrate moiety may also improve site-specific transport to different tissues, which would enhance the peptide bioavailability. On this basis, we have functionalized DCar with ß-cyclodextrin (CDDCar) and characterized the compound via NMR. The copper(II) binding properties of the new DCar derivative were investigated by spectroscopic techniques (UV-vis, circular dichroism, electron paramagnetic resonance) and potentiometric measurements. The results surprisingly revealed a pronounced difference from the analogous LCar derivative (CDLCar), especially concerning the dimeric species. The spectroscopic data show that this stereoselectivity is driven by noncovalent interactions, namely, hydrogen bonds, CH-π interactions, and steric and hydrophobic effects of the cyclodextrin cavity.

Copper complexes of synthetic peptides mimicking neurotrophin-3 enhance neurite outgrowth and CREB phosphorylation.

In this work we report on the synthesis and physiochemical/biological characterization of a peptide encompassing the first thirteen residues of neurotrophin-3 (NT-3). The protein capability to promote neurite outgrowth and axonal branching by a downstream mechanism that involves the increase of the cAMP response element-binding level (CREB) was found for the NT3(1-13) peptide, thus validating its protein mimetic behaviour. Since copper ions are also involved in neurotransmission and their internalization may be an essential step in neuron differentiation and CREB phosphorylation, the peptide and its copper complexes were characterized by potentiometric and spectroscopic techniques, including UV-visible, CD and EPR. To have a detailed picture of the coordination features of the copper complexes with NT3(1-13), we also scrutinized the two peptide fragments encompassing the shorter sequences 1-5 and 5-13, respectively, showing that the amino group is the main anchoring site for Cu(ii) at physiological pH. The peptide activity increased in the presence of copper ions. The effect of copper(ii) addition is more marked for NT3(1-13) than the other two peptide fragments, in agreement with its higher affinity for metal ions. Confocal microscopy measurements carried out on fluorescently labelled NT3(1-13) indicated that copper ions increase peptide internalization.

Multitarget trehalose-carnosine conjugates inhibit Aß aggregation, tune copper(II) activity and decrease acrolein toxicity.

Increasing evidence is accumulating, showing that neurodegenerative disorders are somehow associated with the toxicity of amyloid aggregates, metal ion dyshomeostasis as well as with products generated by oxidative stress. Within the biological oxidation products, acrolein does have a prominent role. A promising strategy to deal with the above neurogenerative disorders is to use multi-functions bio-molecules. Herein, we show how a class of bio-conjugates takes advantage of the antiaggregating, antioxidant and antiglycating properties of trehalose and carnosine. Their ability to sequester acrolein and to inhibit both self- and metal-induced aggregation is here reported. The copper(II) coordination properties of a new trehalose-carnosine conjugate and the relative antioxidant effects have also been investigated.