Chelator PBT2 Forms a Ternary Cu2+ Complex with ß-Amyloid That Has High Stability but Low Specificity.

The metal chelator PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) acts as a terdentate ligand capable of forming binary and ternary Cu2+ complexes. It was clinically trialed as an Alzheimer's disease (AD) therapy but failed to progress beyond phase II. The ß-amyloid (Aß) peptide associated with AD was recently concluded to form a unique Cu(Aß) complex that is inaccessible to PBT2. Herein, it is shown that the species ascribed to this binary Cu(Aß) complex in fact corresponds to ternary Cu(PBT2)NImAß complexes formed by the anchoring of Cu(PBT2) on imine nitrogen (NIm) donors of His side chains. The primary site of ternary complex formation is His6, with a conditional stepwise formation constant at pH 7.4 (Kc [M-1]) of logKc = 6.4 ± 0.1, and a second site is supplied by His13 or His14 (logKc = 4.4 ± 0.1). The stability of Cu(PBT2)NImH13/14 is comparable with that of the simplest Cu(PBT2)NIm complexes involving the NIm coordination of free imidazole (logKc = 4.22 ± 0.09) and histamine (logKc = 4.00 ± 0.05). The 100-fold larger formation constant for Cu(PBT2)NImH6 indicates that outer-sphere ligand-peptide interactions strongly stabilize its structure. Despite the relatively high stability of Cu(PBT2)NImH6, PBT2 is a promiscuous chelator capable of forming a ternary Cu(PBT2)NIm complex with any ligand containing an NIm donor. These ligands include histamine, L-His, and ubiquitous His side chains of peptides and proteins in the extracellular milieu, whose combined effect should outweigh that of a single Cu(PBT2)NImH6 complex regardless of its stability. We therefore conclude that PBT2 is capable of accessing Cu(Aß) complexes with high stability but low specificity. The results have implications for future AD therapeutic strategies and understanding the role of PBT2 in the bulk transport of transition metal ions. Given the repurposing of PBT2 as a drug for breaking antibiotic resistance, ternary Cu(PBT2)NIm and analogous Zn(PBT2)NIm complexes may be relevant to its antimicrobial properties.

Intermediate Cu(II)-Thiolate Species in the Reduction of Cu(II)GHK by Glutathione: A Handy Chelate for Biological Cu(II) Reduction.

Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV-vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.

Ternary Cu2+ Complexes of Human Serum Albumin and Glycyl-l-histidyl-l-lysine.

Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu2+ as part of their normal functions. GHK is found at its highest concentration in the albumin-rich fraction of plasma, leading to speculation that HSA and GHK form a ternary Cu2+ complex. Although preliminary evidence was presented 40 years ago, the structure and stability of such a complex have remained elusive. Here, we show that two ternary Cu(GHK)NImHSA complexes are formed between GHK and the imino nitrogen (NIm) of His side chains of HSA. We identified His3 as one site of ternary complex formation (conditional binding constant cKCu(GHK)NImHis3Cu(GHK) = 2900 M-1 at pH 7.4), with the second site (cKCu(GHK)NImHisXCu(GHK) = 1700 M-1) likely being supplied by either His128 or His510. Together with the established role of HSA as a molecular shuttle in the blood, these complexes may aid the transport of the exchangeable Cu2+ pool and the functional form of GHK.

The Palladium(II) Complex of Aß4-16 as Suitable Model for Structural Studies of Biorelevant Copper(II) Complexes of N-Truncated Beta-Amyloids.

The Aß4-42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer's Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in Aß4-16 and Aß4-9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)Aß4-x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-Aß4-16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)Aß4-16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of Aß4-16, Pd(II)-Aß4-16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-Aß complexes.

Aldehyde Production as a Calibrant of Ultrasonic Power Delivery During Protein Misfolding Cyclic Amplification.

The protein misfolding cyclic amplification (PMCA) technique employs repeated cycles of incubation and sonication to amplify minute amounts of misfolded protein conformers. Spontaneous (de novo) prion formation and ultrasonic power level represent two potentially interrelated sources of variation that frustrate attempts to replicate results from different laboratories. We previously established that water splitting during PMCA provides a radical-rich environment leading to oxidative damage to substrate molecules as well as the polypropylene PCR tubes used for sample containment. Here it is shown that the cross-linking agent formaldehyde is generated from buffer ions that are attacked by hydroxyl radicals. In addition, free radical damage to protein, nucleic acid, lipid, and detergent molecules produces a substantial concentration of aldehydes (hundreds of micromolar). The measurement of aldehydes using the Hantzsch reaction provides a reliable and inexpensive method for measuring the power delivered to individual PMCA samples, and for calibrating the power output characteristics of an individual sonicator. The proposed method may also be used to better account for inter-assay and inter-laboratory variation in prion replication and de novo prion generation, the latter of which may correlate with aldehyde-induced cross-linking of substrate molecules.

Ternary Cu(II) Complex with GHK Peptide and Cis-Urocanic Acid as a Potential Physiologically Functional Copper Chelate.

The tripeptide NH2-Gly-His-Lys-COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.

The Sub-picomolar Cu2+ Dissociation Constant of Human Serum Albumin.

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+ -binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, circular dichroism and room-temperature electron paramagnetic resonance spectroscopies, together with potentiometric titrations, we competed the tripeptide GGH against HSA to reveal a conditional binding constant of log cKCuCu(HSA) =13.02±0.05 at pH 7.4. This rigorously determined value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.

Identification of the Binding Site of Apical Membrane Antigen†1 (AMA1) Inhibitors Using a Paramagnetic Probe.

Apical membrane antigen 1 (AMA1) is essential for the invasion of host cells by malaria parasites. Several small-molecule ligands have been shown to bind to a conserved hydrophobic cleft in Plasmodium falciparum AMA1. However, a lack of detailed structural information on the binding pose of these molecules has hindered their further optimisation as inhibitors. We have developed a spin-labelled peptide based on RON2, the native binding partner of AMA1, to probe the binding sites of compounds on PfAMA1. The crystal structure of this peptide bound to PfAMA1 shows that it binds at one end of the hydrophobic groove, leaving much of the binding site unoccupied and allowing fragment hits to bind without interference. In paramagnetic relaxation enhancement (PRE)-based NMR screening, the 1 H relaxation rates of compounds binding close to the probe were enhanced. Compounds experienced different degrees of PRE as a result of their different orientations relative to the spin label while bound to AMA1. Thus, PRE-derived distance constraints can be used to identify binding sites and guide further hit optimisation.

Oligopeptides Generated by Neprilysin Degradation of ß-Amyloid Have the Highest Cu(II) Affinity in the Whole Aß Family.

The catabolism of ß-amyloid (Aß) is carried out by numerous endopeptidases including neprilysin, which hydrolyzes peptide bonds preceding positions 4, 10, and 12 to yield Aß4-9 and a minor Aß12- x species. Alternative processing of the amyloid precursor protein by ß-secretase also generates the Aß11- x species. All these peptides contain a Xxx-Yyy-His sequence, also known as an ATCUN or NTS motif, making them strong chelators of Cu(II) ions. We synthesized the corresponding peptides, Phe-Arg-His-Asp-Ser-Gly-OH (Aß4-9), Glu-Val-His-His-Gln-Lys-am (Aß11-16), Val-His-His-Gln-Lys-am (Aß12-16), and pGlu-Val-His-His-Gln-Lys-am (pAß11-16), and investigated their Cu(II) binding properties using potentiometry, and UV-vis, circular dichroism, and electron paramagnetic resonance spectroscopies. We found that the three peptides with unmodified N-termini formed square-planar Cu(II) complexes at pH 7.4 with analogous geometries but significantly varied Kd values of 6.6 fM (Aß4-9), 9.5 fM (Aß12-16), and 1.8 pM (Aß11-16). Cyclization of the N-terminal Glu11 residue to the pyroglutamate species pAß11-16 dramatically reduced the affinity (5.8 nM). The Cu(II) affinities of Aß4-9 and Aß12-16 are the highest among the Cu(II) complexes of Aß peptides. Using fluorescence spectroscopy, we demonstrated that the Cu(II) exchange between the Phe-Arg-His and Val-His-His motifs is very slow, on the order of days. These results are discussed in terms of the relevance of Aß4-9, a major Cu(II) binding Aß fragment generated by neprilysin, as a possible Cu(II) carrier in the brain.

The N-terminal 14-mer model peptide of human Ctr1 can collect Cu(ii) from albumin. Implications for copper uptake by Ctr1.

Human cells acquire copper primarily via the copper transporter 1 protein, hCtr1. We demonstrate that at extracellular pH 7.4 CuII is bound to the model peptide hCtr11-14via an ATCUN motif and such complexes are strong enough to collect CuII from albumin, supporting the potential physiological role of CuII binding to hCtr1.

Structural Insight into Redox Dynamics of Copper Bound N-Truncated Amyloid-ß Peptides from in Situ X-ray Absorption Spectroscopy.

X-ray absorption spectroscopy of CuII amyloid-ß peptide (Aß) under in situ electrochemical control (XAS-EC) has allowed elucidation of the redox properties of CuII bound to truncated peptide forms. The Cu binding environment is significantly different for the Aß1-16 and the N-truncated Aß4-9, Aß4-12, and Aß4-16 (Aß4-9/12/16) peptides, where the N-truncated sequence (F4R5H6) provides the high-affinity amino-terminal copper nickel (ATCUN) binding motif. Low temperature (ca. 10 K) XAS measurements show the adoption of identical CuII ATCUN-type binding sites (CuIIATCUN) by the first three amino acids (FRH) and a longer-range interaction modeled as an oxygen donor ligand, most likely water, to give a tetragonal pyramid geometry in the Aß4-9/12/16 peptides not previously reported. Both XAS-EC and EPR measurements show that CuII:Aß4-16 can be reduced at mildly reducing potentials, similar to that of CuII:Aß1-16. Reduction of peptides lacking the H13H14 residues, CuII:Aß4-9/12, require far more forcing conditions, with metallic copper the only metal-based reduction product. The observations suggest that reduction of CuIIATCUN species at mild potentials is possible, although the rate of reduction is significantly enhanced by involvement of H13H14. XAS-EC analysis reveals that, following reduction, the peptide acts as a terdentate ligand to CuI (H13, H14 together with the linking amide oxygen atom). Modeling of the EXAFS is most consistent with coordination of an additional water oxygen atom to give a quasi-tetrahedral geometry. XAS-EC analysis of oxidized CuII:Aß4-12/16 gives structural parameters consistent with crystallographic data for a five-coordinate CuIII complex and the CuIIATCUN complex. The structural results suggest that CuII and the oxidation product are both accommodated in an ATCUN-like binding site.

Interplay between Copper, Neprilysin, and N-Truncation of ß-Amyloid.

Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the ß-amyloid (Aß) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aß levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aß fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP ( Ki = 1.0 µM),  while proteolysis of Aß yielded the soluble, Aß4-9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aß4-9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations ( Ki = 20 µM) further suggests a mechanism for modulating NEP activity, Aß4-9 production, and Cu2+ homeostasis.

Prion protein cleavage fragments regulate adult neural stem cell quiescence through redox modulation of mitochondrial fission and SOD2 expression.

Neurogenesis continues in the post-developmental brain throughout life. The ability to stimulate the production of new neurones requires both quiescent and actively proliferating pools of neural stem cells (NSCs). Actively proliferating NSCs ensure that neurogenic demand can be met, whilst the quiescent pool makes certain NSC reserves do not become depleted. The processes preserving the NSC quiescent pool are only just beginning to be defined. Herein, we identify a switch between NSC proliferation and quiescence through changing intracellular redox signalling. We show that N-terminal post-translational cleavage products of the prion protein (PrP) induce a quiescent state, halting NSC cellular growth, migration, and neurite outgrowth. Quiescence is initiated by the PrP cleavage products through reducing intracellular levels of reactive oxygen species. First, inhibition of redox signalling results in increased mitochondrial fission, which rapidly signals quiescence. Thereafter, quiescence is maintained through downstream increases in the expression and activity of superoxide dismutase-2 that reduces mitochondrial superoxide. We further observe that PrP is predominantly cleaved in quiescent NSCs indicating a homeostatic role for this cascade. Our findings provide new insight into the regulation of NSC quiescence, which potentially could influence brain health throughout adult life.

The Cu(II) affinity of the N-terminus of human copper transporter CTR1: Comparison of human and mouse sequences.

Copper Transporter 1 (CTR1) is a homotrimeric membrane protein providing the main route of copper transport into eukaryotic cells from the extracellular milieu. Its N-terminal extracellular domain, rich in His and Met residues, is considered responsible for directing copper into the transmembrane channel. Most of vertebrate CTR1 proteins contain the His residue in position three from N-terminus, creating a well-known Amino Terminal Cu(II)- and Ni(II)-Binding (ATCUN) site. CTR1 from humans, primates and many other species contains the Met-Asp-His (MDH) sequence, while some rodents including mouse have the Met-Asn-His (MNH) N-terminal sequence. CTR1 is thought to collect Cu(II) ions from blood copper transport proteins, including albumin, but previous reports indicated that the affinity of N-terminal peptide/domain of CTR1 is significantly lower than that of albumin, casting serious doubt on this aspect of CTR1 function. Using potentiometry and spectroscopic techniques we demonstrated that MDH-amide, a tripeptide model of human CTR1 N-terminus, binds Cu(II) with K of 1.3 × 1013 M-1 at pH 7.4, ~13 times stronger than Human Serum Albumin (HSA), and MNH-amide is even stronger, K of 3.2 × 1014 M-1 at pH 7.4. These results indicate that the N-terminus of CTR1 may serve as intermediate binding site during Cu(II) transfer from blood copper carriers to the transporter. MDH-amide, but not MNH-amide also forms a low abundance complex with non-ATCUN coordination involving the Met amine, His imidazole and Asp carboxylate. This species might assist Cu(II) relay down the peptide chain or its reduction to Cu(I), both steps necessary for the CTR1 function.

Electron paramagnetic resonance microscopy using spins in diamond under ambient conditions.

Magnetic resonance spectroscopy is one of the most important tools in chemical and bio-medical research. However, sensitivity limitations typically restrict imaging resolution to ~ 10 µm. Here we bring quantum control to the detection of chemical systems to demonstrate high-resolution electron spin imaging using the quantum properties of an array of nitrogen-vacancy centres in diamond. Our electron paramagnetic resonance microscope selectively images electronic spin species by precisely tuning a magnetic field to bring the quantum probes into resonance with the external target spins. This provides diffraction limited spatial resolution of the target spin species over a field of view of 50 × 50 µm2 with a spin sensitivity of 104 spins per voxel or ∼100 zmol. The ability to perform spectroscopy and dynamically monitor spin-dependent redox reactions at these scales enables the development of electron spin resonance and zepto-chemistry in the physical and life sciences.Electron paramagnetic resonance spectroscopy has important scientific and medical uses but improving the resolution of conventional methods requires cryogenic, vacuum environments. Simpson et al. show nitrogen vacancy centres can be used for sub-micronmetre imaging with improved sensitivity in ambient conditions.

In Vivo-Near Infrared Imaging of Neurodegeneration.

In vivo near-infrared (NIR) imaging of molecular processes at the preclinical stage promises to provide more valuable mechanistic information about pathological pathways involved in neurodegeneration. NIR imaging has the potential to improve in vivo therapeutic screening protocols by enabling noninvasive monitoring of presymptomatic responses to treatment. We have developed new NIR fluorescent contrast agents conjugated to markers of cell death, and using these agents we have identified molecular pathways associated with prion-induced neurodegeneration and determined the optimal window for meaningful therapeutic intervention in prion disease. This chapter provides a description of the synthesis and purification of our NIR cell Death (NIRD) contrast agent and the application of in vivo NIRD (iNIRD) imaging to a prion model of neurodegeneration.

Systematically Studying the Effect of Fluoride on the Properties of Cyclophanes Bearing Naphthalene Diimide and Dialkoxyaryl Groups.

Anion-π interactions between the Lewis basic anion fluoride and π-acidic naphthalene diimide was systematically studied in a series of cyclophanes in which the properties are modulated through the influence of a second, electron-rich aromatic unit. The systems and subsequently generated radical anions, upon addition of fluoride, were studied by absorption spectroscopic and EPR techniques. The results infer a modulation as a result of the nature and strength of the π-π interaction in the macrocyclic structure.

The Case for Abandoning Therapeutic Chelation of Copper Ions in Alzheimer's Disease.

The "therapeutic chelation" approach to treating Alzheimer's disease (AD) evolved from the metals hypothesis, with the premise that small molecules can be designed to prevent transition metal-induced amyloid deposition and oxidative stress within the AD brain. Over more than 20 years, countless in vitro studies have been devoted to characterizing metal binding, its effect on Aß aggregation, ROS production, and in vitro toxicity. Despite a lack of evidence for any clinical benefit, the conjecture that therapeutic chelation is an effective approach for treating AD remains widespread. Here, the author plays the devil's advocate, questioning the experimental evidence, the dogma, and the value of therapeutic chelation, with a major focus on copper ions.

Copper Exchange and Redox Activity of a Prototypical 8-Hydroxyquinoline: Implications for Therapeutic Chelation.

The N-truncated ß-amyloid (Aß) isoform Aß4-x is known to bind Cu(2+) via a redox-silent ATCUN motif with a conditional Kd = 30 fM at pH 7.4. This study characterizes the Cu(2+) interactions and redox activity of Aßx-16 (x = 1, 4) and 2-[(dimethylamino)-methyl-8-hydroxyquinoline, a terdentate 8-hydroxyquinoline (8HQ) with a conditional Kd(CuL) = 35 pM at pH 7.4. Metal transfer between Cu(Aß1-16), CuL, CuL2, and ternary CuL(NIm(Aß)) was rapid, while the corresponding equilibrium between L and Aß4-16 occurred slowly via a metastable CuL(NIm(Aß)) intermediate. Both CuL and CuL2 were redox-silent in the presence of ascorbate, but a CuL(NIm) complex can generate reactive oxygen species. Because the NIm(Aß) ligand will be readily exchangeable with NIm ligands of ubiquitous protein His side chains in vivo, this class of 8HQ ligand could transfer Cu(2+) from inert Cu(Aß4-x) to redox-active CuL(NIm). These findings have implications for the use of terdentate 8HQs as therapeutic chelators to treat neurodegenerative disease.