N4-Tetradentate chelators efficiently regulate copper homeostasis and prevent ROS production induced by copper-amyloid-\xce\xb21-16, even in the presence of an excess of zinc
The disruption of copper homeostasis and the oxidative stress induced by Cu-amyloids are crucial features of Alzheimer\xe2\x80\x99s disease pathology. The copper specific N4-tetradendate ligands TDMQ20 and 1 are able to fully inhibit in vitro the aerobic oxidation of ascorbate induced by Cu-A\xce\xb21-16, even in the presence of 100 molar equivalents of Zn(II) with respect to Cu(II), while other ligands with N2O2 or N3O2 coordination spheres failed to do so. This essential result indicates that, in addition to metal selectivity, the coordination sphere of copper chelators should exhibit a N4-tetradendate motif to be able to reduce an oxidative stress in the zinc-rich physiological environment of brain. The N4-scaffolds of these two aminoquinoline-based ligands, TDMQ20 or 1, suitable for a square-planar coordination of copper(II), allowed them to enhance both the selectivity for copper and also the ability to reduce the oxidative stress induced by copper-amyloid in a zinc-rich environment.
<p>Redistribution and restoration of the homeostasis of metal ions, especially copper, in the brain of patients with Alzheimerâs disease (AD) is now considered as a valuable challenge in the chemotherapy of this neurodegenerative condition.1 However, zinc element is abundant and strictly regulated in the brain to avoid depletion or excess.2 In addition to zinc-protein complexes, large amounts of chelatable zinc are located in the cerebral cortex and the limbic region, notably in the hippocampal formation, and concentrated within synaptic vesicles of glutamatergic neurons where it acts as a modulator of synaptic transmission.2c Zinc concentration has been estimated to reach 300 Î¼M in the synaptic cleft system during strong stimulation.2,3 The mean copper concentration in human frontal lobe and cerebellum has been reported to be in the range of 60-110 Î¼M.4 The zinc/copper ratio can be as high as 10 to 100, in glutamatergic neurons.5a Therefore, any attempt to regulate copper homeostasis and to inhibit copper-induced oxidative stress in the brain, must take into account this zinc-rich environment.5b</p><p>Therefore, the ligands designed to regulate the homeostasis of redox metal ions in AD brains must be copper specific to avoid the perturbation of zinc. As an example of the importance of the possible copper/zinc competition, clioquinol has been discarded due to its neurotoxicity attributed to zinc chelation.6 As a matter of fact, the metal selectivity of 8-hydroxyquinoline scaffold is very low.7 To regulate copper homeostasis, chelators should be able to transfer copper from its pathological sink, mainly from the copper-amyloid complexes (Cu-AÎ²), to glutathione which is a physiological provider of copper to copper enzymes,8 even in the presence of zinc. In addition, a large excess of zinc should not prevent the ability of these copper chelators to inhibit the production of reactive oxygen species (ROS) generated by copper loaded amyloids. Moreover, such ligands should have logP values expected for membrane crossing, especially the blood brain barrier (BBB).</p><p>However, among the copper chelators which are currently under investigation, the selectivity for copper with respect to zinc is usually not (or poorly) documented,1a except for bis-8-aminoquinoline derivatives that are specific copper chelators.9 In this view, some of us reported the capacity of a water-soluble negatively charged chelator named L2 (Figure 1) to remove copper from copper-loaded AÎ² in the presence of zinc in order to illustrate the importance of the Cu selectivity on ROS formation.5 This N2O2 chelator, based on a Schiff-base scaffold, was able to inhibit the reductive activation of dioxygen in the presence of 1 molar equivalent (mol equiv) of Zn(II) but failed to stop ROS production in the presence of 5 mol equiv of Zn(II) during the ascorbate consumption.5a In addition, as pointed out in the article, the ligand L2 is not able to cross the BBB (logP = â 3.7).</p><p>To enhance the efficiency and drugability of copper chelators, we designed a new series of neutral N4-tetradendate copper ligands named TDMQ, based on an 8-aminoquinoline motif.10 One of these ligands, TDMQ20 (Figure 1), is a specific ligand for copper compared to zinc. In the present work, we investigated the ability of TDMQ20 to inhibit the Cu-AÎ² activation of dioxygen in the presence of a large excess of zinc(II), up to 100 mol equiv. The extraction of copper from Cu-AÎ² by TDMQ20, as well as the release of copper from Cu-TDMQ20 in the presence of glutathione, were also evaluated in the presence of zinc, as these two discrete steps are required to put back copper into normal physiological circulation. The obtained data were compared with that of ligand 1, a bis-8-aminoquinoline chelator previously reported to have a very high selectivity for Cu(II) with respect to Zn(II) {log [Kapp Cu-L / Kapp Zn-L] > 12}, and to efficiently transfer copper from Cu-AÎ² to glutathione in the absence of zinc.9 The 8-hydroxyquinoline derivative PBT21c,11 was used as a comparator. A detailed experimental section is provided as Supporting Information.</p><p>The ability of TDMQ20 to extract copper from Cu-AÎ²1-16 was evaluated as follows. The Cu(II) complex of AÎ²1-16 was prepared by incubation of AÎ²1-16 and Cu2+ at 20 Î¼M (molar ratio = 1/1) in Hepes buffer pH 7.4. One or three mol equiv of Zn2+ were added into the CuII-AÎ²1-16 solution. One mol equiv of TDMQ20 was then added, and the reaction was monitored by UV-visible spectroscopy. The spectrum of the resulting mixture containing AÎ²1-16, Cu(II), Zn(II) and TDMQ20 was recorded (Figure 2, red trace, full line for 1 equiv of Zn, dashed line for 3 equiv of Zn). In both cases, the spectrum exhibited absorbance at 243 nm assigned to Cu-TDMQ20 (bright blue trace), but not at 259 and 342 nm, specific wavelengths of the TDMQ20 free ligand. In fact, the spectra of the mixture CuII-AÎ²1-16 / TDMQ20 in the presence of Zn2+ (red traces) were very similar to the arithmetic addition of the spectra of free AÎ²1-16 and Cu-TDMQ20 (black dashed trace). This result is the same as that obtained in a reaction carried out in the absence of zinc (Figure S1, Supporting Information). It therefore clearly indicates that 1 mol equiv of TDMQ20 was sufficient to fully demetalate the copper loaded amyloid, providing Cu-TDMQ20, in the presence of either 1 or 3 mol equiv of zinc with respect to copper. These data indicate that the presence of 3 mol equiv of zinc does not interfere with the demetalation of CuII-AÎ²1-16 by one equivalent of TDMQ20.</p><p>The release of copper from CuII-TDMQ20 in the presence of GSH was then monitored. As previously reported, when the Cu(II) complex of the tetradendate ligand 1 was treated by glutathione (GSH), GSH acts both as a reducing agent of Cu(II) and as a competitive ligand for the generated Cu(I).9c The resulting Cu(I)-glutathione complex is a physiological supplier of copper to the apo form of copper proteins.8 However, the role of zinc in this process has not been reported yet. Consequently, the influence of zinc on copper circulation in the presence of chelators remained to be established. We decided to monitor, by UV-visible spectrometry, the demetalation of CuII-TDMQ20 in the presence of both glutathione and Zn2+, in Hepes buffer, pH 7.4. Firstly, CuCl2 was incubated with TDMQ20 to generate CuII-TDMQ20, and 1 mol equiv of ZnCl2 was then added. The spectrum of the reaction mixture exhibited a maximal absorbance at 243 nm, typical of that expected for CuII-TDMQ20 (Figure 3, red trace). Upon addition of glutathione (by aliquots of 20 mol equiv with respect to Cu-TDMQ), the demetalation of Cu-TDMQ was monitored by formation of the free ligand TDMQ20 detected at 259 and 342 nm (well-defined isosbestic points are detected at 279 and 332 nm; no isosbestic point can be detected below 250 nm, due to the own absorbance of glutathione and Cu(I)-glutathione at these wavelengths) (Figure 3). The plotting of the absorbance at 259 nm as a function of the number of glutathione equivalents (Figure 3, Insert) indicate that 80 equiv of glutathione were required to achieve full demetalation of Cu-TDMQ. Interestingly, a similar result was observed in the absence of ZnCl2 (Figure S2, Supporting Information), indicating that the presence of zinc did not influence the demetalation of Cu-TDMQ20 by glutathione in these conditions.</p><p>So, in vitro, TDMQ20 is able to abstract copper from Cu-AÎ²1-16 and then to transfer it to glutathione, indicating that this N4-tetradendate ligand has the capacity for regulation of copper homeostasis in the presence of zinc.</p><p>The in vitro ROS production mediated by Cu-AÎ²1-16 in the presence or in the absence of any additive (namely zinc or a putative chelator) can be evaluated based on the kinetics of ascorbate oxidation, easily monitored by UV-visible at 265 nm.9c,12,13 We investigated the influence of TDMQ20 (and ligands 1 and PBT2 as comparators, structures in Figure 1) on the oxidation of ascorbate induced by Cu-AÎ²1-16 in the presence of Zn2+. Firstly, a Zn/Cu molar ratio equal to 3 was investigated since senile plaques contain roughly 3 times more Zn than Cu (25 and 69 Î¼g/g for Cu and Zn, respectively).14 The results are showed in Figure 4. Trace (a) shows the ascorbate autoxidation, without any additive. In the presence of CuCl2, ascorbate was fully oxidized in only 5 min (trace b). Cu-AÎ²1-16 induced ca. 40% of ascorbate oxidation in 5 min, and full oxidation occurred in ca. 25 min (trace c). In the presence of 3 mol equiv of Zn2+, TDMQ20 or ligand 1 drastically inhibited the ascorbate oxidation induced by Cu-AÎ²1-16, the oxidation of ascorbate in 30 min being only 15-16% in both cases (traces d and e, respectively), very close to the ascorbate autoxidation (8%, trace a).</p><p>We also performed similar experiments with PBT2, a bi- or tridentate ligand based on 8-hydroxyquinoline scaffold, that has been developed as a potential AD drug.1c,15 Since the Cu-PBT2 complex has potentially a metal/ligand stoichiometry = 1/2,16 we therefore used 2.2 mol equiv of PBT2 with respect to Cu-AÎ²1-16, to avoid the presence of free Cu2+ ions in the solution. With 3 mol equiv of Zn with respect to copper, 89% of ascorbate oxidation was observed in 30 min (Figure 4, trace f), close to the 80% value reported in the absence of zinc9c, indicating that PBT2 did not inhibit ascorbate oxidation under these reaction conditions.</p><p>Since both TMDQ20 and compound 1 efficiently inhibited the ascorbate oxidation induced by Cu-AÎ²1-16 in the presence of 3 mol equiv of Zn2+, we decided to investigate the same reaction with a very large excess of zinc, 100 mol equiv of zinc with respect to copper, a Zn/Cu ratio that has been proposed to be biologically relevant.5 In these conditions, Cu-AÎ²1-16 induced 48% of ascorbate oxidation in 5 min, and 86% in 10 min (Figure 5, trace a). This is a moderate increase of ROS production compared to the result obtained in the absence of zinc (trace b, 39% and 63% in 5 min and 10 min, respectively). A previous report indicated that a single zinc equivalent had no effect on Cu-AÎ² induced ROS production, even though the coordination of Zn2+ impacts the Cu(II) and Cu(I) binding sites of AÎ², and may lead to the formation of a hetero-bimetallic Cu-Zn-AÎ² complex, without release of Cu.17</p><p>Our result indicates that a 100-fold excess of Zn2+ resulted in the release of a fraction of Cu2+ from CuII-AÎ²1-16, which is consistent with a higher ascorbate oxidation. So, the presence of a local huge excess of zinc with respect to Cu-AÎ² may significantly exacerbate the redox toxicity of Cu-AÎ² in vivo.</p><p>In order to evidence the fast action of such N4-tetradendate ligands on the kinetics of the ascorbate oxidation induced by CuCl2/AÎ²1-16/ZnCl2 (1/1/100 molar ratio), we added one of these copper chelators, TDMQ20 or 1 (1 mol equiv), after 6.6 min of reaction (Figure 5, traces d and e, respectively). In both cases, the oxidation of ascorbate was immediately and completely stopped, indicating that stoichiometric amounts of TDMQ20 and 1 were able to fully inhibit the ROS production induced by the mixture Cu(II)/AÎ²1-16/Zn(II) = 1/1/100. The slopes of traces d and e after 6.6 min (ÎAbs265 = 0.002 / min) were not significantly different to that of ascorbate autoxidation (Figure 4, trace a, ÎAbs265 = 0.003 / min).</p><p>These tetradendate ligands TDMQ20 and 1, that specifically chelate Cu(II) in a N4-square planar fashion, are able to immediately and completely stop the in vitro ongoing aerobic oxidation of ascorbate induced by Cu-AÎ²1-16, even in the presence of 100 mol equiv of Zn(II) with respect to copper ions, while the N2O2-tetradentate ligand L2, able to chelate copper with metal/ligand stoichiometry = 1/1, was efficient only in the presence of a stoichiometric amount of zinc.5 PBT2, whose copper complex has a N3O2 coordination sphere, and a metal/ligand stoichiometry = 1/2,16 failed to inhibit oxygen reduction either in the presence or in the absence of zinc.</p><p>These N4-tetradendate ligands TDMQ20 and 1 have a high selectivity for Cu(II) with respect to Zn(II) {log [Kapp Cu-L / Kapp Zn-L] > 12},9a,10 much higher than that of AÎ² {log [Kapp Cu-AÎ² / Kapp Zn-AÎ²] = 4-5},5b and thus fulfill the criteria for biologically pertinent copper chelators mentioned in the introduction. The obtained results indicated that both ligands TDMQ20 and 1 have the potential to reduce or inhibit in vivo ROS production induced by Cu-AÎ², even in a very rich zinc physiological environment.</p><p>In conclusion, such N4-tetradentate copper-specific ligands, like TDMQ20 and 1, are suitable for a square-planar scaffold with Cu(II) and have the ability to largely stabilize Cu(II) over Cu(I). This structural feature makes them efficient inhibitors of the in vitro ROS production induced by Cu-AÎ², including in a zinc-rich physiological environment. Finally, the calculated logP values are 2.5 and 3.5, for TDMQ20 and 1,18 respectively, in the optimal range expected for crossing the blood-brain barrier.19 The design of copper ligands able to counteract metal deregulation and deleterious consequences in AD brain requires a good understanding of their coordination properties. Pharmacological studies on TDMQ20 are in progress.</p>