Thermodynamics-based rules of thumb to evaluate the interaction of chelators and kinetically-labile metal ions in blood serum and plasma.

Metal ions play a very important role in nature and their homeostasis is crucial. A lot of metal-related chemical research activities are ongoing that concern metal-based drugs or tools, such as chelation therapy, metal- and metabolite sensors, metallo-drugs and prodrugs, PET and MRI imaging agents, etc. In most of these cases, the applied chelator/ligand (L) or metal-ligand complex (M-L) has at least to pass the blood plasma to reach the target. Hence it is exposed to several metal-binding proteins (mainly serum albumin and transferrin) and to all essential metal ions (zinc, copper, iron, etc.). This holds also for studies in cultured cells when fetal calf serum is used in the medium. There is a risk that the applied compound (L or M-L) in the serum is transformed into a different entity, due to trans-metallation and/or ligand exchange reactions. This depends on the thermodynamics and kinetics. For kinetically-labile complexes, the complex stability with all the ligands and all metal ions present in serum is decisive in evaluating the thermodynamic driving force towards a certain fate of the chelator or metal-ligand complex. To consider that, an integrative view is needed on the stability constants, by taking into account all the metal ions present and all the main proteins to which they are bound, as well as the non-occupied metal binding site in proteins. Only then, a realistic estimation of the complex stability, and hence its potential fate, can be done. This perspective aims to provide a simple approach to estimate the thermodynamic stability of labile metal-ligand complexes in a blood plasma/serum environment. It gives a guideline to obtain an estimation of the plasma and serum complex stability and metal selectivity starting from the chemical stability constants of metal-ligand complexes. Although of high importance, it does not focus on the more complex kinetic aspects of metal-transfer reactions. The perspective should help for a better design of such compounds, to perform test tube assays which are relevant to the conditions in the plasma/serum and to be aware of the importance of ternary complexes, kinetics and competition experiments.

Cu(II) Binding to the N-Terminal Model Peptide of the Human Ctr2 Transporter at Lysosomal and Extracellular pH.

It was shown that His3 of human copper transporter 1 (hCtr1) prompts the ATCUN-like Cu(II) coordination for model peptides of the hCtr1 N-terminus. Its high Cu(II) affinity is a potential driving force for the transfer of Cu(II) from extracellular Cu(II) carriers to hCtr1. Having a sequence similar to that of hCtr1, hCtr2 has been proposed as another human copper transporter. However, the N-terminal domain of hCtr2 is much shorter than that of hCtr1, with different copper binding motifs at its N-terminus. Employing a model peptide of the hCtr2 N-terminus, MAMHF-am, we demonstrated that His4 provides a unique pattern of Cu(II) complexes, involving Met sulfurs in their Cu(II) coordination sphere. The affinity of Cu(II) for MAMHF-am is a few orders of magnitude lower than that reported for the hCtr1 model peptides at the extracellular pH of 7.4, suggesting a maximal complementary role of Cu(II) binding to hCtr2 in the import of copper from the extracellular space to the cytoplasm. On the other hand, the ability of the hCtr2 model peptide to capture Cu(II) from amino acids and short peptides (potential degradation products of proteins) at pH 5.0 and the known predominant lysosomal localization of hCtr2 support an important potential role of the Cu(II)-hCtr2 interaction in the recovery of copper from lysosomes.

Identification of key structural features of the elusive Cu-Aß complex that generates ROS in Alzheimer's disease.

Oxidative stress is linked to the etiology of Alzheimer's disease (AD), the most common cause of dementia in the elderly. Redox active metal ions such as copper catalyze the production of Reactive Oxygen Species (ROS) when bound to the amyloid-ß (Aß) peptide encountered in AD. We propose that this reaction proceeds through a low-populated Cu-Aß state, denoted the "catalytic in-between state" (CIBS), which is in equilibrium with the resting state (RS) of both Cu(i)-Aß and Cu(ii)-Aß. The nature of this CIBS is investigated in the present work. We report the use of complementary spectroscopic methods (X-ray absorption spectroscopy, EPR and NMR) to characterize the binding of Cu to a wide series of modified peptides in the RS. ROS production by the resulting Cu-peptide complexes was evaluated using fluorescence and UV-vis based methods and led to the identification of the amino acid residues involved in the Cu-Aß CIBS species. In addition, a possible mechanism by which the ROS are produced is also proposed. These two main results are expected to affect the current vision of the ROS production mechanism by Cu-Aß but also in other diseases involving amyloidogenic peptides with weakly structured copper binding sites.

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.

Copper coordination to native N-terminally modified versus full-length amyloid-ß: second-sphere effects determine the species present at physiological pH.

Alzheimer's disease is characterized by senile plaques in which metallic ions (copper, zinc, and iron) are colocalized with amyloid-ß peptides of different sequences in aggregated forms. In addition to the full-length peptides (Aß1-40/42), N-terminally truncated Aß3-40/42 forms and their pyroglutamate counterparts, Aßp3-40/42, have been proposed to play key features in the aggregation process, leading to the senile plaques. Furthermore, they have been shown to be more toxic than the full-length Aß, which made them central targets for therapeutic approaches. In order to better disentangle the possible role of metallic ions in the aggregation process, copper(II) coordination to the full-length amyloid peptides has been extensively studied in the last years. However, regarding the N-terminally modified forms at position 3, very little is known. Therefore, copper(I) and copper(II) coordination to those peptides have been investigated in the present report using a variety of complementary techniques and as a function of pH. Copper(I) coordination is not affected by the N-terminal modifications. In contrast, copper(II) coordination is different from that previously reported for the full-length peptide. In the case of the pyroglutamate form, this is due to preclusion of N-terminal amine binding. In the case of the N-terminally truncated form, alteration in copper(II) coordination is caused by second-sphere effects that impact the first binding shell and the pH-dependent repartition of the various [Cu(peptide)] complexes. Such second-sphere effects are anticipated to apply to a variety of metal ions and peptides, and their importance on changing the first binding shell has not been fully recognized yet.

Zinc binding to amyloid-beta: isothermal titration calorimetry and Zn competition experiments with Zn sensors.

Aggregation of the peptide amyloid-beta (Abeta) to amyloid plaques is a key event in Alzheimer's disease. According to the amyloid cascade hypothesis, Abeta aggregates are toxic to neurons via the production of reactive oxygen species and are hence directly involved in the cause of the disease. Zinc ions play an important role, because they are able to bind to Abeta and influence the aggregation properties. In the present work isothermal titration calorimetry and Zn sensors (zincon, Newport Green, and zinquin) were used to investigate the interaction of Zn with the full-length Abeta1-40 and Abeta1-42, as well as the truncated Abeta1-16 and Abeta1-28. The results suggest that Zn binding to Abeta induces a release of approximately 0.9 proton by the peptide. This correspond to the expected value upon Zn binding to the three histidines and indicates that further ligands are not deprotonated upon Zn binding. Such behavior is expected for carboxylates, but not the N-terminus. Moreover, the apparent dissociation constant (Kd,app) of Zn binding to all forms of Abeta is in the low micromolar range (1-20 microM) and rather independent of the aggregation state including soluble Abeta, Abeta fibrils, or Zn-induced Abeta aggregates. Finally, Zn in the soluble or aggregated Zn-Abeta form is well accessible for Zn chelators. The potential repercussions on metal chelation therapy are discussed.

Redox silencing of copper in metal-linked neurodegenerative disorders: reaction of Zn7metallothionein-3 with Cu2+ ions.

Dysregulation of copper and zinc homeostasis in the brain plays a critical role in Alzheimer disease (AD). Copper binding to amyloid-beta peptide (Abeta) is linked with the neurotoxicity of Abeta and free radical damage. Metallothionein-3 (MT-3) is a small cysteine- and metal-rich protein expressed in the brain and found down-regulated in AD. This protein occurs intra- and extracellularly, and it plays an important role in the metabolism of zinc and copper. In cell cultures Zn7MT-3, by an unknown mechanism, protects neurons from the toxicity of Abeta. We have, therefore, used a range of complementary spectroscopic and biochemical methods to characterize the interaction of Zn7MT-3 with free Cu2+ ions. We show that Zn7MT-3 scavenges free Cu2+ ions through their reduction to Cu+ and binding to the protein. In this reaction thiolate ligands are oxidized to disulfides concomitant with Zn2+ release. The binding of the first four Cu2+ is cooperative forming a Cu(I)4-thiolate cluster in the N-terminal domain of Cu4,Zn4MT-3 together with two disulfides bonds. The Cu4-thiolate cluster exhibits an unusual stability toward air oxygen. The results of UV-visible, CD, and Cu(I) phosphorescence at 77 K suggest the existence of metal-metal interactions in this cluster. We have demonstrated that Zn7MT-3 in the presence of ascorbate completely quenches the copper-catalyzed hydroxyl radical (OH.) production. Thus, zinc-thiolate clusters in Zn7MT-3 can efficiently silence the redox-active free Cu2+ ions. The biological implication of our studies as to the protective role of Zn7MT-3 from the Cu2+ toxicity in AD and other neurodegenerative disorders is discussed.