In order to mitigate superoxide-induced damage, cells rely on superoxide dismutases (SODs), a family of metalloenzymes that catalyze the dismutation of superoxide. These enzymes display a positively-charged substrate entrance channel for superoxide guidance to the active metal center. ATCUN-like Ni(II) complexes have been shown to mimic NiSOD effectively and benefit from versatile peptide structures, which are ideal for introducing positive charges through arginine residues at various positions to modulate catalytic properties. We describe here five novel ATCUN-like Ni(II) complexes, with arginines at various positions in close proximity to the metal center. All the peptides form monometallic square planar Ni(II) complexes in water at pH 7.4, with either a N2S2 coordination (S-series) or an equilibrium between N2S2 and N3S1 coordination modes (N/S-series). The S-series demonstrates a clear beneficial effect of the charge increase from − 2 to + 2 on the pseudo-first order catalytic constant. Furthermore, the data obtained for the N/S complexes demonstrate that introducing an arginine residue in the central position of the ATCUN-like motif does not enhance activity, but actually impedes catalysis. These findings show that both the distance of the charged residues from the metal center and their positioning in space are of prime importance.

Maintaining tightly copper homeostasis is crucial for the survival of all living organisms, in particular microorganisms like bacteria. They have evolved a number of proteins to capture, transport and deliver Cu(I), while avoiding Fenton-like reactions. Some Cu proteins exhibit methionine-rich (Met-rich) domains, whose role remains elusive. In this work, we designed biomimetic compounds recapitulating the possible Cu(I) binding sites in these domains, in order to examine the parameters important for Cu(I) binding. Five different biomimetic pseudopeptides were synthesized, exhibiting either three methionines or two methionines and a third amino acid likely to be present in the Met-rich domain. The affinities for Cu(I) of these model binding sites were determined, as well as their redox properties and behavior in the presence of Cu(II). Our results highlight the importance of Met residues, and their abundance in Met-rich domains, to efficiently bind Cu(I) in the periplasmic space.

In the field of nuclear toxicology, the knowledge of the interaction of actinides (An) with biomolecules is of prime concern in order to elucidate their toxicity mechanism and to further develop selective decorporating agents. In this work, we demonstrated the great potential of hydrophilic interaction liquid chromatography (HILIC) to separate polar thorium (Th) biomimetic peptide complexes, as a key starting point to tackle these challenges. Th4+ was used as plutonium (Pu4+) analogue and pS16 and pS1368 as synthetic di- and tetraphosphorylated peptides capable of mimicking the interaction sites of these An in osteopontin (OPN), a hyperphosphorylated protein. The objective was to determine the relative affinity of pS16 and pS1368 towards Th4+, and to evaluate the pS1368 selectivity when Th4+ 4 + was in competition complexation reaction with UO 2 at physiological pH. To meet these aims, HILIC was simultaneously coupled to electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS), which allowed to identify online the molecular structure of the separated complexes and quantify them, in a single step. Dedicated HILIC conditions were firstly set up to separate the new dimeric Th2(peptide)2 2 (peptide) 2 complexes with good separation resolution (peptide = pS16 or pS1368). By adding pS16 and pS1368 in different proportions relatively to Th4+, 4 + , we found that lower or equal proportions of pS16 with respect to pS1368 were not sufficient to displace pS1368 from Th2pS13682 2 pS1368 2 and pS16 proportion higher than pS1368 led to the formation of a predominant ternary complex Th2(pS16)(pS1368), 2 (pS16)(pS1368), demonstrating preferential Th4+ 4 + binding to the tetra-phosphorylated peptide. Finally, online identification and quantification of the formed complexes when Th4+ 4 + and UO 2 2 + were mixed in equimolar ratio relatively to pS1368 showed that in spite of pS1368 has been specifically designed to coordinate UO22+, pS1368 is also Th4+-selective 4 +-selective and exhibits stronger affinity for this latter than for UO22+. Hence, the results gathered through this approach highlight the impact of Th4+ coordination chemistry on its interaction with pS1368 and more widely to its affinity for biomolecules.

We present here the most active synthetic Ni superoxide dismutase (NiSOD) mimic reported to date. Reactive oxygen species are aggressive compounds, which concentrations are tightly regulated in vivo. Among them, the superoxide anion, O 2 ⋅ − , is controlled by superoxide dismutases. Capitalizing on the versatility of the Amino-Terminal Cu II - and Ni II -binding (ATCUN) peptide motif, we introduced positive charges around the Ni II center to favor the interaction with the superoxide radical anion. At physiological pH, the pentapeptide H−Cys−His−Cys−Arg−Arg−NH 2 coordinates Ni II after the deprotonation of one thiol, two amides, and either the second thiol or the N-terminal ammonium, leading to an equilibrium between the two N 3 S 1 and N 2 S 2 coordination modes. Under catalytic conditions, a k cat value of 8.6(4)×10 6  L.mol −1 .s −1 was measured. Within the first second, the catalyst remained undegraded with quantitative consumption of O 2 ⋅ − (completed up to 37 catalytic cycles). An extra arginine (Arg) was introduced at the peptide C-terminus to increase the global charge of the Ni II complex from +1 to +2. This had no effect on the catalytic performance, highlighting the critical role of charge distribution in space as a determining factor influencing the reactivity.

The AsIII binding of two NTA-based tripodal pseudopeptides, possessing three cysteine (ligand L1) or d-penicillamine residues (ligand L2) as potential coordinating groups for soft semimetals or metal ions, was studied by experimental (UV, CD, NMR, and ESI-MS) and theoretical (DFT) methods. All of the experimental data, obtained with the variation of the AsIII:ligand concentration ratios or pH values in some instances, evidence the exclusive formation of species with an AsS3-type coordination mode. The UV-monitored titration of the ligands with arsenous acid at pH = 7.0 provided an absorbance data set that allowed for the determination of apparent stability constants of the forming species. The obtained stabilities (logK' = 5.26 (AsL1) and logK' = 3.04 (AsL2)) reflect high affinities, especially for the sterically less restricted cysteine derivative. DFT calculated structures correlate well with the spectroscopic results and, in line with the 1H NMR data, indicate a preference for the all-endo conformers resembling the AsIII environment at the semimetal binding sites in various metalloproteins.

Several proteins have been identified in the past decades as targets of uranyl (UO22+) in vivo. However, the molecular interactions responsible for this affinity are still poorly known which requires the identification of the UO22+ coordination sites in these proteins. Biomimetic peptides are efficient chemical tools to characterize these sites. In this work, we developed a dedicated analytical method to determine the affinity of biomimetic, synthetic, multi-phosphorylated peptides for UO22+ and evaluate the effect of several structural parameters of these peptides on this affinity at physiological pH. The analytical strategy was based on the implementation of the simultaneous coupling of hydrophilic interaction chromatography (HILIC) with electrospray ionization mass spectrometry (ESI-MS) and inductively coupled plasma mass spectrometry (ICP-MS). An essential step had been devoted to the definition of the best separation conditions of UO22+ complexes formed with di-phosphorylated peptide isomers and also with peptides of different structure and degrees of phosphorylation. We performed the first separations of several sets of UO22+ complexes by HILIC ever reported in the literature. A dedicated method had then been developed for identifying the separated peptide complexes online by ESI-MS and simultaneously quantifying them by ICP-MS, based on uranium quantification using external calibration. Thus, the affinity of the peptides for UO22+ was determined and made it possible to demonstrate that (i) the increasing number of phosphorylated residues (pSer) promotes the affinity of the peptides for UO22+, (ii) the position of the pSer in the peptide backbone has very low impact on this affinity (iii) and finally the cyclic structure of the peptide favors the UO22+ complexation in comparison with the linear structure. These results are in agreement with those previously obtained by spectroscopic techniques, which allowed to validate the method. Through this approach, we obtained essential information to better understand the mechanisms of toxicity of UO22+ at the molecular level and to further develop selective decorporating agents by chelation.

The cellular level of reactive oxygen species (ROS) has to be controlled to avoid some pathologies, especially those linked to oxidative stress. One strategy for designing antioxidants consists of modeling natural enzymes involved in ROS degradation. Among them, nickel superoxide dismutase (NiSOD) catalyzes the dismutation of the superoxide radical anion, O2•-, into O2 and H2O2. We report here Ni complexes with tripeptides derived from the amino-terminal CuII- and NiII-binding (ATCUN) motif that mimics some structural features found in the active site of the NiSOD. A series of six mononuclear NiII complexes were investigated in water at physiological pH with different first coordination spheres, from compounds with a N3S to N2S2 set, and also complexes that are in equilibrium between the N-coordination (N3S) and S-coordination (N2S2). They were fully characterized by a combination of spectroscopic techniques, including 1H NMR, UV-vis, circular dichroism, and X-ray absorption spectroscopy, together with theoretical calculations and their redox properties studied by cyclic voltammetry. They all display SOD-like activity, with a kcat ranging between 0.5 and 2.0 × 106 M-1 s-1. The complexes in which the two coordination modes are in equilibrium are the most efficient, suggesting a beneficial effect of a nearby proton relay.

Uranium (U) is a naturally-occurring radionuclide that is toxic to living organisms. Given that proteins are primary targets of U(VI), their identification is an essential step towards understanding the mechanisms of radionuclide toxicity, and possibly detoxification. Here, we implemented a chromatographic strategy including immobilized metal affinity chromatography to trap protein targets of uranyl in Arabidopsis thaliana. This procedure allowed the identification of 38 uranyl-binding proteins (UraBPs) from root and shoot extracts. Among them, UraBP25, previously identified as plasma membrane-associated cation-binding protein 1 (PCaP1), was further characterized as a protein interacting in vitro with U(VI) and other metals using spectroscopic and structural approaches, and in planta through analyses of the fate of U(VI) in Arabidopsis lines with altered PCaP1 gene expression. Our results showed that recombinant PCaP1 binds U(VI) in vitro with affinity in the nM range, as well as Cu(II) and Fe(III) in high proportions, and that Ca(II) competes with U(VI) for binding. U(VI) induces PCaP1 oligomerization through binding at the monomer interface, at both the N-terminal structured domain and the C-terminal flexible region. Finally, U(VI) translocation in Arabidopsis shoots was affected in pcap1 null-mutant, suggesting a role for this protein in ion trafficking in planta.

A cyclic tetra-phosphorylated biomimetic peptide (pS1368) has been proposed as a promising starting structure to design a decorporating agent of uranyl (UO22+) due to its affinity being similar to that of osteopontin (OPN), a target UO22+ protein in vivo. The determination of this peptide’s selectivity towards UO22+ in the presence of competing endogenous elements is also crucial to validate this hypothesis. In this context, the selectivity of pS1368 towards UO22+ in the presence of Ca2+, Cu2+ and Zn2+ was determined by applying the simultaneous coupling of hydrophilic interaction chromatography (HILIC) to electrospray ionization (ESI–MS) and inductively coupled plasma (ICP-MS) mass spectrometry. Sr2+ was used as Ca2+ simulant, providing less challenging ICP-MS measurements. The separation of the complexes by HILIC was first set up. The selectivity of pS1368 towards UO22+ was determined in the presence of Sr2+, by adding several proportions of the latter to UO2(pS1368). UO22+ was not displaced from UO2(pS1368) even in the presence of a ten-fold excess of Sr2+. The same approach has been undertaken to demonstrate the selectivity of pS1368 towards UO22+ in the presence of Cu2+, Zn2+ and Sr2+ as competing endogenous cations. Hence, we showed that pS1368 was selective towards UO22+ in the presence of Sr2+, but also in the presence of Cu2+ and Zn2+. This study highlights the performance of HILIC-ESI-MS/ICP-MS simultaneous coupling to assess the potential of molecules as decorporating agents of UO22+.

As an alpha emitter and chemical toxicant, uranium toxicity in living organisms is driven by its molecular interactions. It is therefore essential to identify main determinants of uranium affinity for proteins. Others and we showed that introducing a phosphoryl group in the coordination sphere of uranyl confers a strong affinity of proteins for uranyl. In this work, using calmodulin site 1 as a template, we modulate the structural organization of a metal-binding loop comprising carboxylate and/or carbonyl ligands and reach affinities for uranyl comparable to that provided by introducing a strong phosphoryl ligand. Shortening the metal binding loop of calmodulin site 1 from 12 to 10 amino acids in CaMΔ increases the uranyl-binding affinity by about 2 orders of magnitude to log KpH7 = 9.55 ± 0.11 (KdpH7 = 280 ± 60 pM). Structural analysis by FTIR, XAS, and molecular dynamics simulations suggests an optimized coordination of the CaMΔ-uranyl complex involving bidentate and monodentate carboxylate groups in the uranyl equatorial plane. The main role of this coordination sphere in reaching subnanomolar dissociation constants for uranyl is supported by similar uranyl affinities obtained in a cyclic peptide reproducing CaMΔ binding loop. In addition, CaMΔ presents a uranyl/calcium selectivity of 107 that is even higher in the cyclic peptide.

Determining the affinity of proteins for uranyl is key to understand the toxicity of this cation and to further develop decorporation strategies. However, usual techniques to achieve that goal often require specific equipment and expertise. Here, we propose a simple, efficient, fluorescence-based method to assess the affinity of proteins and peptides for uranyl, at equilibrium and in buffered solution. We first designed and characterized an original uranyl-binding fluorescent probe. We then built a reference scale for uranyl affinity in solution, relying on signal quenching of our fluorescent probe in presence of high-affinity uranyl-binding peptides. We finally validated our approach by re-evaluating the uranyl-binding affinity of four native proteins. We envision that this tool will facilitate the reliable and reproducible assessment of affinities of peptides and proteins for uranyl.

Liver is the main organ for metabolism but is also subject to various pathologies, from viral, genetic, cancer or metabolic origin. There is thus a crucial need to develop efficient liver-targeted drug delivery strategies. Asialoglycoprotein receptor (ASGPR) is a C-type lectin expressed in the hepatocyte plasma membrane that efficiently endocytoses glycoproteins exposing galactose (Gal) or N-acetylgalactosamine (GalNAc). Its targeting has been successfully used to drive the uptake of small molecules decorated with three or four GalNAc, thanks to an optimisation of their spatial arrangement. Herein, we assessed the biological properties of highly stable nanostructured lipid carriers (NLC) made of FDA-approved ingredients and formulated with increasing amounts of GalNAc. Cellular studies showed that a high density of GalNAc was required to favour hepatocyte internalisation via the ASGPR pathway. Interaction studies using surface plasmon resonance and the macrophage galactose-lectin as GalNAc-recognising lectin confirmed the need of high GalNAc density for specific recognition of these NLC. This work is the first step for the development of efficient nanocarriers for prolonged liver delivery of active compounds.

Imidazole thiones appear as interesting building blocks for Cu(I) chelation and protection against Cu-mediated oxidative stress. Therefore, a series of tripodal molecules derived from nitrilotriacetic acid appended with three imidazole thiones belonging either to histamine-like or histidine-like moieties were synthesized. These tripods demonstrate intermediate affinity between that previously measured for tripodal analogues bearing three thiol moieties such as cysteine and those grafted with three thioethers, like methionines, consistently with the thione group in the imidazole thione moiety existing as a tautomer between a thiol and a thione. The two non-alkylated tripods derived from thioimidazole, TH and TH* demonstrated three orders of magnitude larger affinity for Cu(I) (logKpH 7.4 = 14.3) than their analogues derived from N,N'-dialkylated thioimidazole TMe and TEt (logKpH 7.4 = 11-11.6). Their efficiency to inhibit Cu-mediated oxidative stress is demonstrated by several assays involving ascorbate consumption or biomolecule damages and correlates with their ability to chelate Cu(I), related to their conditional complexation constants at pH 7.4. The two non-alkylated tripods derived from thioimidazole, TH and TH* are significantly more powerful in reducing Cu-mediated oxidative stress than their analogues derived from N,N'-dialkylated thioimidazole TMe and TEt.

Copper homeostasis is finely regulated in human to avoid any detrimental impact of free intracellular copper ions. Upon copper accumulation, biliary excretion is triggered in liver thanks to trafficking of the ATP7B copper transporter to bile canaliculi. However, in Wilson's disease this protein is mutated leading to copper accumulation. Current therapy uses Cu chelators acting extracellularly and requiring a life-long treatment with side effects. Herein, a new Cu(I) pro-chelator was encapsulated in long-term stable nanostructured lipid carriers. Cellular assays revealed that the pro-chelator protects hepatocytes against Cu-induced cell death. Besides, the cellular stresses induced by moderate copper concentrations, including protein unfolding, are counteracted by the pro-chelator. These data showed the pro-chelator efficiency to deliver intracellularly an active chelator that copes with copper stress and surpasses current and under development chelators. Although its biological activity is more mitigated, the pro-chelator nanolipid formulation led to promising results. This innovative approach is of outmost importance in the quest of better treatments for Wilson's disease.

Peptides are efficient models used in different fields such as toxicology to study the interactions of several contaminants at the molecular scale, requiring the development of bio-analytical strategies. In this context, Hydrophilic interaction liquid chromatography (HILIC) coupled to electrospray ionization mass spectrometry (ESI-MS) was used to separate synthetic multiphosphorylated cyclopeptides and their positional isomers at physiological pH. We assessed (i) the selectivity of eleven HILIC columns, from different manufacturers and packed with diverse polar sorbents, and (ii) the effect of mobile phase composition on the separation selectivity. The best selectivity and baseline resolution were achieved with the columns grafted by neutral sorbents amide and diol. Furthermore, we investigated the HILIC retention mechanism of these peptides by examining the effect of the number of phosphorylated residues in the peptide scaffold on their retention. The peptide behavior followed the classical hydrophilic partitioning mechanism exclusively on amide and diol columns. This trend was not fully respected on bare and hybrid silica due to the attractive/repulsive interactions of the deprotonated surface silanol groups with the Arginine or Glutamate residues in the peptide scaffold according to the peptide sequence. The position of the phosphorylated amino acid in the peptide backbone also showed to have an impact on the retention, making possible the separation of positional isomers of these multiphosphorylated cyclic peptides using HILIC.

Nickel superoxide dismutase (NiSOD) is an enzyme that protects cells against O2·-. While the structure of its active site is known, the mechanism of the catalytic cycle is still not elucidated. Its active site displays a square planar NiII center with two thiolates, the terminal amine and an amidate. We report here a bioinspired NiII complex built on an ATCUN-like binding motif modulated with one cysteine, which demonstrates catalytic SOD activity in water (kcat = 8.4(2) × 105 M-1 s-1 at pH = 8.1). Its reactivity with O2·- was also studied in acetonitrile allowing trapping two different short-lived species that were characterized by electron paramagnetic resonance or spectroelectrochemistry and a combination of density functional theory (DFT) and time-dependent DFT calculations. Based on these observations, we propose that O2·- interacts first with the complex outer sphere through a H-bond with the peptide scaffold in a [NiIIO2·-] species. This first species could then evolve into a NiIII hydroperoxo inner sphere species through a reaction driven by protonation that is thermodynamically highly favored according to DFT calculations.

Inhalation of (Co3O4)-Co-60 particles may occur at the work place in nuclear industry. Their low solubility may result in chronic lung exposure to gamma rays. Our strategy for an improved therapeutic approach is to enhance particle dissolution to facilitate cobalt excretion, as the dissolved fraction is rapidly eliminated, mainly in urine. In vitro dissolution of Co3O4 particles was assessed with two complementary assays in lung fluid surrogates to mimic a pulmonary contamination scenario. Twenty-one molecules and eleven combinations were selected through an extensive search in the literature, based on dissolution studies of other metal oxides (Fe, Mn, Cu) and tested for dissolution enhancement of cobalt particles after 1-28 days of incubation. DTPA, the recommended treatment following cobalt contamination did not enhance (Co3O4)-Co-60 particles dissolution when used alone. However, by combining molecules with different properties, such as redox potential and chelating ability, we greatly improved the efficacy of each drug used alone, leading for the highest efficacy, to a 2.7 fold increased dissolution as compared to controls. These results suggest that destabilization of the particle surface is an important initiating event for a good efficacy of chelating drugs, and open new perspectives for the identification of new therapeutic strategies.

Uranium is an element belonging to the actinide series. It is ubiquitous in rock, soil, and water. Uranium is found in the ecosystem due to mining and milling industrial activities and processing to nuclear fuel, but also to the extensive use of phosphate fertilizers. Understanding uranium binding in vivo is critical, first to deepen our knowledge of molecular events leading to chemical toxicity, but also to provide new mechanistic information useful for the development of efficient decorporation treatments to be applied in case of intoxication. The most stable form in physiological conditions is the uranyl cation (UO22+), in which uranium oxidation state is +VI. This short review presents uranyl coordination properties and chelation, and what is currently known about uranium binding to proteins. Although several target proteins have been identified, the UO22+ binding sites have barely been identified. Biomimetic approaches using model peptides are good options to shed light on high affinity uranyl binding sites in proteins. A strategy based on constrained cyclodecapeptides allowed recently to propose a tetraphosphate binding site for uranyl that provides an affinity similar to the one measured with the phosphoprotein osteopontin.

Grafting thiol-bearing molecules at the surface of silver nanoparticles (AgNPs) is a successful strategy to tune their optical and antibacterial properties. The capping layer generated from self-assembly of the ligands at the nanoparticle surface determines the range of possible applications of the resulting material. In particular, direct grafting of the thiol heads to surface Ag(I) can occur, with various hybridizations of the S atom, sp versus sp(3). Alternatively, a passivating Ag2S layer can form. We make use of S K-edge X-ray absorption near edge structure (XANES) and synchrotron-based X-ray photoelectron spectroscopy (XPS) to probe the metal-ligand interface in different thiol-capped AgNPs. The use of cryogenic conditions for XAS analyses reveals a peculiar spectral signature for thiolates chemisorbed on the AgNPs surface, unambiguously distinguished from that of Ag2S. Ab initio simulations of XANES spectra and XPS analyses are used to predict the grafting mode, suggesting that different ligand architectures promote slightly different proportions of sp/sp(3) sites, and a dramatic variability in the stability of the nanomaterial that can evolve toward either self-assembly or dissolution of the AgNPs.