Modulation of Amyloid-β Aggregation by Histidine-coordinating Cobalt(III) Schiff Base Complexes

Abstract

Oligomers of the Aβ42 peptide are significant neurotoxins linked to Alzheimer’s Disease (AD). Histidine (His) residues present at the N-terminus of Aβ42 are believed to influence toxicity by either serving as metal-ion binding sites (that promote oligomerization and oxidative damage) or facilitating synaptic binding. Transition metal complexes that bind to these residues and modulate Aβ toxicity have emerged as therapeutic candidates. Cobalt(III) Schiff base complexes (Co(III)-sb) were evaluated for their ability to interact with Aβ peptides. HPLC-MS, NMR, fluorescence, and DFT studies demonstrated that Co(III)-sb complexes could interact with the His residues in a truncated Aβ16 peptide representing the Aβ42 N-terminus. Coordination of Co(III)-sb complexes altered the structure of Aβ42 peptides and promoted the formation of large soluble oligomers. Interestingly, this structural perturbation of Aβ correlated to reduced synaptic binding to hippocampal neurons. These results demonstrate the promise of Co(III)-sb complexes in anti-AD therapeutic approaches.

Alzheimer’s Disease (AD) is a neurodegenerative disorder that manifests as impaired memory formation and cognitive decline.^[1] Progression of this devastating disease has been linked to the accumulation of oligomers of amyloid-β peptides (Aβ) in the brain.^[2] Aβ42 peptides form toxic aggregates that can target neuronal synapse and cause detrimental disruption of receptor populations, morphology, and stability.^{[2d, 2e, 3]} Pathologies induced by Aβ oligomers include tau hyperphosphorylation, reactive oxygen species (ROS) accumulation, synapse deterioration, and ultimately, nerve cell death.^{[2f, 2g, 2k, 4]} Blocking formation of these species is a promising approach for slowing and even preventing AD progression.^{[1a, 2a, 2b, 5]}

While the exact mechanism that governs Aβ-induced dysfunction is widely debated, significant research has demonstrated that the histidines (His) at residues 6, 13, and 14 in the Aβ sequence influence the toxicity of the peptide.^[6] Some researchers have suggested that the His residues form a putative Aβ metal-ion binding site.^{[6c, 6d, 7]} This type of binding activity can sequester endogenous metals (such as Cu²⁺ and Zn²⁺) in Aβ, disrupting normal metalloenzyme activity and catalyzing harmful ROS generation. Studies have additionally suggested that metal-ion binding may stabilize Aβ conformations that mediate the formation of toxic Aβ oligomers and aggregates.^[8] Other work has shown that independent of metal-ion binding, His13 and -14 facilitate aberrant interactions of Aβ peptides with neuronal synapses.^{[6a, 6e, 6f]} While the exact role of the His residues in Aβ toxicity is debated, interfering with these residues is a promising approach to modulating toxicity.

Transitional metal-containing agents that can bind to His residues have been shown to to disrupt AD neuropathology.^[9] The first example was a class of platinum-phenanthroline complexes, including Pt(1,10-phenanthroline)Cl₂ (Pt-phen, Scheme 1B).^[9a] The platinum complexes disrupted Aβ aggregation and reduced ROS generation through His-coordination. Importantly, these complexes inhibited neurotoxicity and rescued long-term potentiation. Subsequently, ruthenium-, iridium-, and rhodium-based Aβ binding agents were reported that exhibited similar promise for therapeutic development.^[9c–e]

Scheme 1.

(A) Amyloid β (Aβ) peptides used in these studies. Aβ16 was used a soluble peptide model to understand the interactions between Co(III)-sb complexes and the N-terminus of the Aβ peptide. Aβ42 was used to understand the effects of Co(III)-sb complexes on peptide oligomerization and synaptic binding. (B) Transition metal complexes and naming scheme used in this chapter. Co-acacen and Co-benacen are Co(III)-sb complexes that coordinate His residues through dissociative ligand exchange of the axial ammines. The behavior of Co(III)-sb on modulating Aβ was compared to Pt-phen, a Pt(II) complex previously shown to disrupt Aβ-induced neurotoxicity. (C) Proposed scheme of the modulation of Aβ activity by Co-acacen. Co-acacen is believed to coordinate to the His residues of Aβ through the two axial positions. Computational studies suggest the simultaneous coordination of His6 and either His13 or His14 as the most stable conformation. His-coordination alters the Aβ structure, disrupting oligomerization pathways and synaptic binding.

Cobalt(III) Schiff base (Co(III)-sb) complexes inhibit metalloproteins by binding essential His residues through a dissociative ligand exchange at the axial positions.^[10] Hiscoordination is selective over other amino acids such cysteine and lysine.^[11] These complexes consist of an acetylacetonatoethylenediimine (acacen) Schiff base equatorial ligand that stabilizes the cobalt(III) center. Incorporation of a peptide or oligonucleotide targeting moiety confers selectivity for the targets over proteins of the same class.^{[10a, 12]} The success of this approach has been demonstrated with Co(III)-sb/DNA conjugates for the selective inhibition of transcription factors relevant to development and disease.^[12a–c] In a similar manner, selective inhibition of the α-thrombin enzyme was achieved through inclusion of a peptidyl targeting domain.^[12d] Therefore, in addition to the ability to bind to His residues these complexes can be designed for targeted therapeutics.

We hypothesized that the binding of Co(III)-sb to the His residues of Aβ could alter the Aβ structure and alter oligomerization pathways. Through modulation of Aβ activity, Co(III)-sb can serve as both an anti-AD therapeutic and a tool for understanding AD pathology. Here, the interaction between Co(III)-sb complexes and Aβ peptides was investigated.

The truncated peptide Aβ16 was used to evaluate the ability of the Co(III)-sb complex, Co-acacen, to bind to the Aβ N-terminus (Scheme 1A). Aβ16 has been previously used in investigations of the putative metal binding site of Aβ peptides, and is believed to comprise the essential residues including the three histidines in the sequence.^{[7b, 9b, 13]} This peptide serves as a soluble model to evaluate small molecule binding without the complication of spontaneous aggregation of the full peptide.^{[7b, 9b, 14]}

Aβ16 was analyzed by ESI-MS to determine the ability of the cobalt complex Co-acacen to bind the N-terminus of the peptide. The spectra of Aβ16 exhibited m/z values (m = 1955.0, z = 2, 3, 4) corresponding to the peptide alone. Upon incubation of the peptide with one molar equivalent of Co-acacen, additional peaks were observed in the mass spectrum corresponding to a 1:1 peptide adduct with Co-acacen (m = 2238.3, observed m/z correspond to z = 2, 3) (SI Figure 1). This result indicates that Co(III) Schiff complexes bind to the Aβ N-terminus.

The incubation mixture was analyzed by HPLC (SI Figure 2, SI Figure 3). The components of the incubation mixture were separated by elution times, monitored by absorbance at 220 nm (for peptide bonds) and 330 nm (for the acacen ligand of the cobalt complex). ESI-MS spectra of the various components were deconvoluted by the m/z of the free peptide, m/z of Co-acacen (without axial ligands), and m/z of the 1:1 peptide/Co-acacen adduct. Three major components were observed corresponding to the free Aβ16, free Co-acacen, and the peptide/Co-acacen adduct ((SI Figure 2, SI Figure 3B) in contrast to the single component corresponding to the free Aβ16 observed in the analysis of the peptide alone (SI Figure 3A). The HPLC chromatogram corroborates that the peptide/Co-acacen adduct observed in the gas phase (direct injection by ESI-MS) exists in solution phase (SI Figure 2). Further, the eluent at the retention time corresponding to the peptide/Co-acacen adduct showed three peaks, indicating that multiple conformations may be present. Since three histidines are present that can potentially coordinate to the Co(III) center at the two axial positions, multiple conformations are possible.

¹H NMR studies were performed with Aβ16 to determine the binding site of Co-acacen in the sequence (Figure 1). The spectra were obtained in buffered D₂O to reduce signals from the exchangeable backbone amides (NH) of the peptide and simplify analysis. Assignments were made and confirmed by ¹H-¹H TOCSY NMR (SI Figure 4). Spectra of Aβ were acquired at increasing concentrations of Co-acacen (Co-acacen:Aβ16 = 0.0 to 4.0) and the aromatic regions of the spectra (6.0–9.0 ppm) were analyzed. In the absence of Co-acacen, peaks corresponding to the CH2 protons of the free His imidazoles are observed (7.9–8.2 ppm). With increasing equivalents of Co-acacen up to 2.0 equivalents, a concentration-dependent reduction of the these free His peaks is observed, accompanied by the emergence of in a new series of peaks between 6.35–6.7 ppm. The frequencies of these new peaks correspond to the resonances of the CH5 protons of His imidazoles coordinated to Co(III)-sb complexes.^[10b] Further, the decrease in integral vaues of the CH2 protons of the free His imidazoles quantitatively corresponds to rise in integral values of the new series of peaks attributed tot he CH5 protons oft he Co^III-coordinated His upon addition of Co-acacen (SI Figure 5).

Figure 1.

¹H NMR of Aβ16 (5mM) with increasing ratios of Co-acacen:Aβ16 from 0.0 – 4.0 in D₂O buffered with 100 mM phosphate buffer to pH 7.0. Spectra were acquired at on a 500 MHz spectrometer at 25 °C after 1h incubations at 37 °C. With increasing equivalents of Co-acacen, a loss of peaks corresponding to the free His CH2 protons (7.9–8.2 ppm) is observed. New peaks are observed between 6.35–6.7 ppm, indicating His-coordination to Co-acacen (Co(III)-His) in a concentration-dependent manner. A complete loss of free His peaks is observed at ≥ 2.0 eq. of Co-acacen.

The resulting spectra exhibit lower resolution than the free peptide, likely due to conformational heterogeneity. Co-acacen can coordinate any one of the three His residues (Scheme 1C). If 1.0 eq Co-acacen is added, free His CH2 proton peaks are still present (as Co-acacen can only coordinate two equivalents of Co-acacen at a time). Complete loss of free His protons are observed at 2.0 equivalents, indicating that all three His residues are coordinated to Co-acacen. Near complete His-coordination at 2.0 equivalents (rather than 3.0 equivalents) implies that Co-acacen is capable of coordinating two His residues simultaneously.

A further increase in concentration of Co-acacen (≥2.0 equivalents) resulted in the appearance of a new set of peaks at 6.4 ppm. While these new peaks are within the region of the expected chemical shifts of Co(III)-coordinated His CH5 protons, the chemical shifts are distinct from the peaks observed at <2.0 equivalents of 1. These observations indicate the presence of more than one species containing His coordinated to [Co(acacen)(L)₂]⁺ (Co(III)-His). The appearance of these new resonances coincides with loss of signal intensity, which correlates to an increase in conformational heterogeneity. Such heterogeneity is expected, given the multiple possible combinations of His₂ coordination to the Co-acacen (His13/His14, His6/His13, His6/His14).

The conformation and geometry of the Co^III center in the Co-acacen/Aβ16 adduct was evaluated by electronic absorption and compared to the Co-acacen alone ([Co(acacen)(NH₃)₂]⁺), a small molecule model of Co-acacen coordinated to two histidines, [Co(acacen)(4MeIm)₂]⁺ where the 4-methylimidazole ligands (4MeIm) mimics the histidine side chain, and the peptide alone (SI Figure 6A, D). At both and 1.0 and 2.0 equivalents of Co-acacen, the resulting adduct exhibits an electronic absorption spectrum that closely resembles [Co(acacen)(4MeIm)₂]⁺, demonstrating the presence of an octahedral [Co(acacen)(His)₂]⁺ structure with the acacen in the equatorial plane and His₂-coordination at the axial positions (See SI Discussion 1 for extended discussion of electronic absorption spectra). These observations are consistent with previous observations with an established target of Co-acacen, zinc finger transcription factors.^[11]

DFT studies were performed to assess the thermodynamically favorable configuration of axial ligand coordination by the three His residues to Co-acacen. To represent possible modes of Aβ binding, several structures of cobalt complexes with varying axial ligands were located. The His13-His14 portion of the sequence was represented as two histidine side chains linked with a peptide bond.^[15] His6 was represented by 4-methylimidazole, a small molecule model of the histidine side chain.^[10c] The relative energies of the structures were compared to determine a preference for bis-coordination (both axial positions are coordinated to imidazoles) or mono-coordination (one axial position is coordinated to an imidazole and the other is coordinated to an aquo ligand) (Figure 2).

Figure 2.

Thermodynamic parameters of Co(III)-sb complexes coordinated to models of His6, 13, and 14. These calculations indicate the favorability of a coordination mode with the His6 and either the His13 or His14 at the axial positions of the Co(III)-sb complex. Simultaneous coordination of the His13 and His14 is the least favorable due to strain induced on the octahedral geometry (SI Figure 7).

All possible conformations of each structure were optimized using the B3LYP density functional^[16] in conjunction with the 6–31+G(d) Pople basis set for all atoms.^{[10c, 15]} Frequency calculations were performed to confirm that the structures were ground-state minima and obtain thermal corrections to the electronic energy. The thermodynamic parameters of the lowest energy conformations were compared (Figure 2). These calculations predict that the most favorable coordination geometry involves the coordination of His6 at one position and either His13 or His14 at the other position. Slightly less favorable is coordination of just one histidine (with little difference in favorability for coordination of His6, or His13/14). The least favored is the simultaneous coordination of His13 and His14. This data supports a resulting mixture of coordination, consistent with the NMR experiments.

The effects of the cobalt complex on oligomerization pathways of the full Aβ42 peptide were evaluated with SDS-PAGE Western blot and silver stain analysis. Western blots were carried out with the conformation-specific antibody, NU2, that recognizes soluble Aβ oligomers (referred to as ADDLs). ADDLs were prepared at 100μM Aβ42 with increasing concentrations of Co-acacen. In comparison to the untreated preparations, Co-acacen promoted the formation of large (>30kDa) SDS-stable oligomers while reducing the small oligomer population in a concentration-dependent manner (Figure 3A and SI Figure 8).

Figure 3.

SDS-PAGE Western blots of Aβ42 oligomerized in the presence of Co-acacen, Co-benacen, and the free ligands. (A) Co-acacen shifts the distribution of SDS-stable Aβ soluble oligomers to higher MW species in a concentration-dependent manner. (B) A similar effect is observed with Co-benacen but to a greater degree, possibly due to the presence of the aromatic residues. This result is confirmed by the slight stabilization of large oligomers with the free Benacen ligand. Such disruption is not observed with the free Acacen ligand.

This phenomenon was analyzed in comparison to treatment with the previously studied platinum complex, Pt-phen. Although Pt-phen was previously evaluated for effects on Aβ plaque formation, its effects on soluble oligomer formation have not been studied. The SDS-PAGE Western blots with the NU2 antibody demonstrate that Pt-phen similarly promotes the formation of the large SDS-stable oligomer population (Figure 4). The similarity with Pt-phen in the effects on Aβ structure demonstrates the promise of Co-acacen in therapeutic development.

Figure 4.

SDS-PAGE (A) silver stain profiles and (B) Western blots of Aβ42 oligomerized in the presence of Co-acacen, Pt-en and Pt-phen. In a similar manner to Co-acacen, Pt-phen shifts the distribution of SDS-stable Aβ soluble oligomers to higher MW species. This is not observed with Pt(ethylenediamine)Cl₂ (Pt-en), indicating the importance of the aromatic scaffold in Aβ structural disruption. The similarity of the effect of Co-acacen on the Aβ oligomerization to Pt-phen underscores the promise of the cobalt complexes for anti-AD therapeutic approaches.

The planar aromatic scaffold contained in the 1,10-phenanthroline ligand of Pt-phen is posited to interact with the aromatic residues (Tyr10, Phe19, or Phe21) of Aβ through π–π stacking interactions. The non-covalent interactions of the ligand 1) targets the Pt^II center to the amino acids in the peptide 2) affects the reactivity of Pt^II center and 2) influences Aβ aggregation since these hydrophobic residues are associated with self-recognition of the peptide that occurs in oligomerization.^[17] This key role of the phenanthroline ligand is supported by lack of Aβ pathway disruption by cisplatin. In addition to non-covalent interactions, the difference in behavior may also result from a difference in binding modes; while Pt-phen coordinates up to two His residues (with preference for His6 and His14), cisplatin predominantly coordinates to Met35.^[17d] Another platinum complex lacking this aromatic scaffold, Pt(ethylenediamine)Cl₂ (Pt-en) was tested for effects on Aβ oligomer formation (Figure 4). Pt-en did not appear to alter oligomer formation, supporting the influential role of the N-heterocyclic aromatic scaffold on Aβ oligomerization pathways.

To evaluate the influence of aromatic scaffolds on Aβ binding a derivative of Co-acacen containing aromatic groups on the equatorial ligand, Co-benacen, was synthesized and tested for effect on Aβ oligomerization. Oligomerization was analyzed by SDS-PAGE Western blot probed with the oligomer-specific monoclonal antibody NU2 (Figure 3B). At the same concentrations as Co-acacen, Co-benacen demonstrated a similar stabilization of the large oligomer (Figure 3B). A slight difference in oligomer distribution is observed, possibly due to interactions between the aromatic phenyl groups of Co-benacen and the hydrophobic pocket of Aβ42. This hypothesis is supported by a slight stabilization of large oligomers in the presence of the free benacen ligand that is not observed with the free acacen ligand (Figure 3B). HPLC-MS analysis of a solution of Aβ16 treated with Co-benacen demonstrated that the complex can effectively interact with the N-terminus of the Aβ42 peptide that comprise the three His residues (SI Figure 3C).

The effect of Co-acacen on synaptic binding of ADDLs was evaluated with hippocampal neurons to correlate alterations in oligomeric structure to Aβ activity in cells. The Co-benacen complex was not used for these studies due to poor solubility. It has been previously determined that oligomers of particular size are able to bind the synapse.^[18] Synaptic attachment can then initiate Aβ-induced neurotoxicity.^[4d] Differentiated hippocampal neurons were treated with oligomers formed with 30 nM Aβ42 in the presence or absence of Co-acacen. Synaptic binding was analyzed by immunolabeling the cells with NU4, a conformation-specific antibody for Aβ oligomers. Treatment with oligomers assembled in the presence of 100 nM of Co-acacen resulted in a marked decrease in overall binding to neurons (SI Figure 9). Specifically, this overall reduction appears to result from reduction in small NU4-sensitive clusters. These results show promise for Co(III) Schiff base complexes to reduce synaptic targeting of pathogenic Aβ oligomers through biochemically altering Aβ structure.

We have demonstrated that cobalt(III) Schiff base complexes can interact with the N-terminus of Aβ peptides and alter their structure and oligomerization propensities. The specific binding of the cobalt complex, Co-acacen, to the Aβ N-terminus was shown with the Aβ16 peptide. Specifically the three histidines in the sequence were identified as the binding site of the cobalt complexes. Computational studies predict the preference for simultaneous coordination of the His6 and either the His13 or His14 imidazoles to the Co(III) center. However, NMR studies indicate the presence of multiple possible binding conformations.

The cobalt complexes altered Aβ oligomerization pathways, stabilizing the formation of large soluble oligomers. A similar effect was observed with Pt-phen for which promising anti-AD activity was previously demonstrated. The similarity in the effects of the cobalt complexes to Pt-phen presents the cobalt complexes as viable candidates for therapeutic development. This conclusion is validated by the reduction of Aβ synaptic binding induced by the cobalt complex, Co-acacen. Additionally, the stabilization of large oligomers by the natural product oleocanthal has been previously correlated to protection from synaptopathological effects of Aβ.^[19]

These studies reveal the potential of using cobalt(III) Schiff base complex as a platform to develop Aβ-binding molecules. Further, this work underscores the promise of metal-based modulators of His-residues for anti-AD applications. Finally, the cobalt complexes can be modified with an Aβ targeting molecule (such as a peptide or small molecule) to introduce selectivity for the Aβ His residues and improve overall potency.