Spectrally Tunable Förster Resonance Energy Transfer-Based Biosensors Using Organic Dye Grafting.

Biosensors based on Förster resonance energy transfer (FRET) have revolutionized cellular biology by allowing the direct measurement of biochemical processes in situ. Many genetically encoded sensors make use of fluorescent proteins that are limited in spectral versatility and that allow few ways to change the spectral properties once the construct has been created. In this work, we developed genetically encoded FRET biosensors based on the chemigenetic SNAP and HaloTag domains combined with matching organic fluorophores. We found that the resulting constructs can display comparable responses, kinetics, and reversibility compared to their fluorescent protein-based ancestors, but with the added advantage of spectral versatility, including the availability of red-shifted dye pairs. However, we also find that the introduction of these tags can alter the sensor readout, showing that careful validation is required before applying such constructs in practice. Overall, our approach delivers an innovative methodology that can readily expand the spectral variety and versatility of FRET-based biosensors.

Unexpected Trends in Copper Removal from Aß Peptide: When Less Ligand Is Better and Zn Helps.

Cu, Zn, and amyloid-ß (Aß) peptides play an important role in the etiology of Alzheimer's disease (AD). Their interaction indeed modifies the self-assembly propensity of the peptide that is at the origin of the deposition of insoluble peptide aggregates in the amyloid plaque, a hallmark found in AD brains. Another even more important fallout of the Cu binding to Aß peptide is the formation of reactive oxygen species (ROS) that contributes to the overall oxidative stress detected in the disease and is due to the redox ability of the Cu ions. Many therapeutic approaches are currently developed to aid fighting against AD, one of them targeting the redox-active Cu ions. Along this research line, we report in the present article the use of a phenanthroline-based peptide-like ligand (L), which is able to withdraw Cu from Aß and redox-silence it in a very stable 4N Cu(II) binding site even in the presence of Zn(II). In addition and in contrast to what is usually observed, the presence of excess of L lessens the searched effect of ROS production prevention, but it is counterbalanced by the co-presence of Zn(II). To explain such unprecedented trends, we proposed a mechanism that involves the redox reaction between Cu(II)L and Cu(I)L2. We thus illustrated (i) how speciation and redox chemistry can weaken the effect of a ligand that would have appeared perfectly suitable if only tested in a 1:1 ratio and on CuAß and (ii) how Zn overcomes the undesired lessening of ROS arrest due to excess of ligand. In brief, we have shown how working in biologically relevant conditions is important for the understanding of all of the reactions at play and this must be taken into consideration for the further rational design of ligands aiming to become drug candidates.

An easy-to-implement combinatorial approach involving an activity-based assay for the discovery of a peptidyl copper complex mimicking superoxide dismutase.

A combinatorial approach using a one-bead-one-compound method and a screening based on a SOD-activity assay was set up for the discovery of an efficient peptidyl copper complex. The complex exhibited good stability constants, suitable redox potentials and excellent intrinsic activity. This complex was further assayed in cells for its antioxidant properties and showed beneficial effects when cells were subjected to oxidative stress.

Insights into Second-Sphere Effects on Redox Potentials, Spectroscopic Properties, and Superoxide Dismutase Activity of Manganese Complexes with Schiff-Base Ligands.

Six Mn-Schiff base complexes, [Mn(X-salpn)]0/+ (salpn = 1,3-bis(sal-ic-ylidenamino)propane, X = H [1], 5-Cl [2], 2,5-F2 [3], 3,5-Cl2 [4], 5-NO2 [5], 3,5-(NO2)2 [6]), were synthesized and characterized in solution, and second-sphere effects on their electrochemical and spectroscopic properties were analyzed. The six complexes catalyze the dismutation of superoxide with catalytic rate constants in the range 0.65 to 1.54 × 106 M-1 s-1 obtained through the nitro blue tetrazolium photoreduction inhibition superoxide dismutases assay, in aqueous medium of pH 7.8. In solution, these compounds possess two labile solvent molecules in the axial positions favoring coordination of the highly nucleophilic O2 •- to the metal center. Even complex 5, [Mn(5-(NO2)salpn) (OAc) (H2O)], with an axial acetate in the solid state, behaves as a 1:1 electrolyte in methanolic solution. Electron paramagnetic resonance and UV-vis monitoring of the reaction of [Mn(X-salpn)]0/+ with KO2 demonstrates that in diluted solutions these complexes behave as catalysts supporting several additions of excess O2 •-, but at high complex concentrations (≥0.75 mM) catalyst self-inhibition occurs by the formation of a catalytically inactive dimer. The correlation of spectroscopic, electrochemical, and kinetics data suggest that second-sphere effects control the oxidation states of Mn involved in the O2 •- dismutation cycle catalyzed by complexes 1-6 and modulate the strength of the Mn-substrate adduct for electron-transfer through an inner-sphere mechanism.

Copper-Targeting Approaches in Alzheimer's Disease: How To Improve the Fallouts Obtained from in Vitro Studies.

According to the amyloid cascade hypothesis, metal ions, mainly Cu and Zn ions, bound to the amyloid-ß (Aß) peptides are implicated in Alzheimer's disease (AD), a widespread neurodegenerative disease. They indeed impact the aggregation pathways of Aß and are involved in the catalytic generation of reactive oxygen species (ROS) that participate in oxidative stress, while Aß aggregation and oxidative stress are regarded as two key events in AD etiology. Cu ions due to their redox ability have been considered to be the main potential therapeutic targets in AD. A considerable number of ligands have been developed in order to modulate the toxicity associated with Cu in this context, via disruption of the Aß-Cu interaction. Among them, small synthetic ligands and small peptide scaffolds have been designed and studied for their ability to remove Cu from Aß. Some of those ligands are able to prevent Cu(Aß)-induced ROS production and can modify the aggregation pathways of Aß in vitro and in cellulo. Examples of such ligands are gathered in this Viewpoint, as a function of their structures and discussed with respect to their properties against Cu(Aß) deleterious fallouts. Nevertheless, the beneficial activities of the most promising ligands detected in vitro and in cellulo have not been transposed to human yet. Some parameters that might explain this apparent contradiction and key concepts to consider for the design of "more" efficient ligands are thus reported and discussed. En passant, this Viewpoint sheds light on the difficulties in comparing the results from one study to another that hamper significant advances in the field.

Kinetics Are Crucial When Targeting Copper Ions to Fight Alzheimer's Disease: An Illustration with Azamacrocyclic Ligands.

Targeting copper ions to either remove or redistribute them is currently viewed as a possible therapeutic strategy in the context of Alzheimer's disease (AD). Thermodynamic parameters, as for instance the copper(II) affinity of the drug candidate or the copper(II) over zinc(II) selectivity, are considered in the design of the drug candidate. In contrast, kinetic factors have been overlooked despite their probable high importance. In the present article, we use a series of azamacrocyclic ligands to demonstrate that kinetic issues must be taken into account when designing copper-targeting drug candidates in the context of AD.

A Metallo Pro-Drug to Target CuII in the Context of Alzheimer's Disease.

Alzheimer's disease and oxidative stress are connected. In the present communication, we report the use of a MnII -based superoxide dismutase (SOD) mimic ([MnII (L)]+ , 1+ ) as a pro-drug candidate to target CuII -associated events, namely, CuII -induced formation of reactive oxygen species (ROS) and modulation of the amyloid-ß (Aß) peptide aggregation. Complex 1+ is able to remove CuII from Aß, stop ROS and prevent alteration of Aß aggregation as would do the corresponding free ligand LH. Using 1+ instead of LH in further biological applications would have the double advantage to avoid the cell toxicity of LH and to benefit from its proved SOD-like activity.

Mutual interference of Cu and Zn ions in Alzheimer's disease: perspectives at the molecular level.

While metal ions such as copper and zinc are essential in biology, they are also linked to several amyloid-related diseases, including Alzheimer's disease (AD). Zinc and copper can indeed modify the aggregation pathways of the amyloid-ß (Aß) peptide, the key component encountered in AD. In addition, the redox active copper ions do produce Reactive Oxygen Species (ROS) when bound to the Aß peptide. While Cu(i) or Cu(ii) or Zn(ii) coordination to the Aß has been extensively studied in the last ten years, characterization of hetero-bimetallic Aß complexes is still scarce. This is also true for the metal induced Aß aggregation and ROS production, for which studies on the mutual influence of the copper and zinc ions are currently appearing. Last but not least, zinc can strongly interfere in therapeutic approaches relying on copper detoxification. This will be exemplified with a biological lead, namely metallothioneins, and with synthetic ligands.

A Trishistidine Pseudopeptide with Ability to Remove Both CuΙ and CuΙΙ from the Amyloid-ß Peptide and to Stop the Associated ROS Formation.

The pseudopeptide L, derived from a nitrilotriacetic acid scaffold and functionalized with three histidine moieties, is reminiscent of the amino acid side chains encountered in the Alzheimer's peptide (Aß). Its synthesis and coordination properties for CuΙ and CuΙΙ are described. L efficiently complex CuΙΙ in a square-planar geometry involving three imidazole nitrogen atoms and an amidate-Cu bond. By contrast, CuΙ is coordinated in a tetrahedral environment. The redox behavior is irreversible and follows an ECEC mechanism in accordance with the very different environments of the two redox states of the Cu center. This is in line with the observed resistance of the CuΙ complex to oxidation by oxygen and the CuΙΙ complex reduction by ascorbate. The affinities of L for CuΙΙ and CuΙ at physiological pH are larger than that reported for the Aß peptide. Therefore, due to its peculiar Cu coordination properties, the ligand L is able to target both redox states of Cu, redox silence them and prevent reactive oxygen species production by the CuAß complex. Because reactive oxygen species contribute to the oxidative stress, a key issue in Alzheimer's disease, this ligand thus represents a new strategy in the long route of finding molecular concepts for fighting Alzheimer's disease.

Link between Affinity and Cu(II) Binding Sites to Amyloid-ß Peptides Evaluated by a New Water-Soluble UV-Visible Ratiometric Dye with a Moderate Cu(II) Affinity.

Being able to easily determine the Cu(II) affinity for biomolecules of moderate affinity is important. Such biomolecules include amyloidogenic peptides, such as the well-known amyloid-ß peptide involved in Alzheimer's disease. Here, we report the synthesis of a new water-soluble ratiometric Cu(II) dye with a moderate affinity (109 M-1 at pH 7.1) and the characterizations of the Cu(II) corresponding complex by X-ray crystallography, EPR, and XAS spectroscopic methods. UV-vis competition was performed on the Aß peptide as well as on a wide series of modified peptides, leading to an affinity value of 1.6 × 109 M-1 at pH 7.1 for the Aß peptide and to a coordination model for the Cu(II) site within the Aß peptide that agrees with the one mostly accepted currently.

How Zn can impede Cu detoxification by chelating agents in Alzheimer's disease: a proof-of-concept study.

The role of Cu and Zn ions in Alzheimer's disease is linked to the consequences of their coordination to the amyloid-ß (Aß) peptide, i.e. to the modulation of Aß aggregation and to the production of Reactive Oxygen Species (ROS), two central events of the so-called amyloid cascade. The role of both ions in Aß aggregation is still controversial. Conversely the higher toxicity of the redox competent Cu ions (compared to the redox inert Zn ions) in ROS production is acknowledged. Thus the Cu ions can be considered as the main therapeutic target. Because Zn ions are present in higher quantity than Cu ions in the synaptic cleft, they can prevent detoxification of Cu by chelators unless they have an unusually high Cu over Zn selectivity. We describe a proof-of-concept study where the role of Zn on the metal swap reaction between two prototypical ligands and the Cu(Aß) species has been investigated by several complementary spectroscopic techniques (UV-Vis, EPR and XANES). The first ligand has a higher Cu over Zn selectivity relative to the one of Aß peptide while the second one exhibits a classical Cu over Zn selectivity. How Zn impacts the effect of the ligands on Cu-induced ROS production and Aß aggregation is also reported.

Zinc(II) Binding Site to the Amyloid-ß Peptide: Insights from Spectroscopic Studies with a Wide Series of Modified Peptides.

The Zn(II) ion has been linked to Alzheimer's disease (AD) due to its ability to modulate the aggregating properties of the amyloid-ß (Aß) peptide, where Aß aggregation is a central event in the etiology of the disease. Delineating Zn(II) binding properties to Aß is thus a prerequisite to better grasp its potential role in AD. Because of (i) the flexibility of the Aß peptide, (ii) the multiplicity of anchoring sites, and (iii) the silent nature of the Zn(II) ion in most classical spectroscopies, this is a difficult task. To overcome these difficulties, we have investigated the impact of peptide alterations (mutations, N-terminal acetylation) on the Zn(Aß) X-ray absorption spectroscopy fingerprint and on the Zn(II)-induced modifications of the Aß peptides' NMR signatures. We propose a tetrahedrally bound Zn(II) ion, in which the coordination sphere is made by two His residues and two carboxylate side chains. Equilibria between equivalent ligands for one Zn(II) binding position have also been observed, the predominant site being made by the side chains of His6, His13 or His14, Glu11, and Asp1 or Glu3 or Asp7, with a slight preference for Asp1.