Tolcapone Interferes With Key Pathological Features in Alzheimer’s Disease

TL;DR

Tolcapone, a drug for Parkinson's disease, may help treat Alzheimer's by reducing amyloid-beta toxicity and metal interactions.

Contribution

The study reveals how tolcapone interacts with amyloid-beta and copper complexes, offering new insights into its neuroprotective mechanisms.

Findings

Tolcapone binds directly to amyloid-beta monomers.

It acts as a radical scavenger and competes with amyloid-beta for copper ion binding.

Tolcapone disrupts metal-amyloid-beta complexes, potentially inhibiting toxic aggregation.

Abstract

Tolcapone, a clinically approved drug for the treatment of Parkinson’s disease as an adjunct therapy, has recently emerged as a potential modulator of amyloid‐β aggregation and toxicity, which are hallmark features of Alzheimer’s disease and are also involved in ocular neurodegenerative disorders, including glaucoma and age‐related macular degeneration. Despite these noteworthy findings, the molecular basis of the interaction between amyloid‐β and tolcapone remains poorly understood, and the mechanisms by which tolcapone affects metal–amyloid‐β species have yet to be explored. In this work, we investigate the binding interactions of tolcapone with both copper‐free amyloid‐β and copper‐associated amyloid‐β complexes, using a combination of techniques including UV–vis spectroscopy, circular dichroism, mass spectrometry, and surface plasmon resonance. The results reveal that tolcapone binds directly to amyloid‐β monomers. Furthermore, in vitro assays confirm the capacity of tolcapone to act as a radical scavenger and to compete with amyloid‐β for the binding of copper ions. Altogether, our findings suggest that tolcapone exerts a multifaceted protective effect, potentially inhibiting toxic metal‐free and metal aggregation pathways by preventing metal coordination to amyloid‐β or disrupting preformed amyloid‐β–metal complexes, thus offering new perspectives to explore and develop its analogs for the treatment of neurodegenerative disorders.

Figures

Chemical structure of tolcapone with the two vicinal hydroxyl groups emphasized in gray. This structural motif closely resembles that of several well‐known natural polyphenols—such as catechin, quercetin, epigallocatechin, apigenin, resveratrol, luteolin, rosmarinic acid, and morin.

Sensorgram of the interaction between varying Tol concentrations (47–750 μM) and immobilized Aβ1‐40 on the CDL sensor chip, showing increasing SPR response (RU) with rising analyte concentrations. Data were fitted to a 1:1 two‐state binding model (red lines).

ESI‐MS spectrum of Cu2+–Tol 1:1.

UV–vis titration of Cu2+ (0–2 mol equiv) into a solution of Tol at pH 7.4 (0.01 m MOPS/dioxane 70:30). CTol = 4.00 × 10−5 M.

TEAC values at 1, 3, and 6 min for Tol in MOPS (5 mM, pH 7.4). TEAC values are the average of four independent trials, and error bars show standard deviations.

Representative kinetics of AA consumption, measured by UV–vis at 265 nm, of Cu2+ alone, Tol alone, and Cu2+ complexes of Tol. Conditions: AA 100 μM, ligand 4.0 μM, Cu2+ 2.0 μM, HEPES 50 mM pH 7.4, 25°C, cuvette path length of 1 cm.

Tables

TABLE 1. ESI‐MS characterization of Tol and its Cu2+ complexes in water.

Sample	Species	Assignment	Calcd. (m/z)	Exp. (m/z)	RI (%) a
Tol	[T+H]+	C14H12NO5	274.07	274.14	4
	[T2+H]+	C28H23N2O10	547.13	547.54	100
	[T2+Na]+	C28H22N2O10Na	569.12	569.56	40
	[T2+K]+	C28H22N2O10K	585.09	585.58	50
Tol/Cu 1:1	[T+H]+	C14H12NO5	274.07	274.14	20
	[Cu2T−4H]+	C14H7NO5Cu2	394.89	394.98	20
	[Cu2T−4H+H2O]+	C14H9NO6Cu2	412.90	412.98	40
	[Cu2T−4H+CH3OH]+	C15H11NO6Cu2	426.92	427.00	38
	[T2+H]+	C28H23N2O10	547.13	547.14	100
	[CuT2]+	C28H22N2O10CuI	609.06	609.09	35

TABLE 2. ESI‐MS characterization of Tol and its Cu2+ complexes in water.

Sample	Species	Assignment	Calcd. (m/z)	Exp. (m/z)	RI (%) a
Aβ16	[Aβ16+5H]5+	C84H124N27O28	391.78	391.88	70
	[Aβ16+4H]4+	C84H123N27O28	489.48	489.60	100
	[Aβ16+3H]3+	C84H122N27O28	652.30	652.66	80
	[Aβ16+2H]2+	C84H121N27O28	977.94	979.04	30
Aβ16–Cu2+	[Aβ16+5H]5+	C84H124N27O28	391.78	391.96	45
	[Aβ16+Cu+3H]5+	C84H122N27O28Cu	403.96	403.98	40
	[Aβ16+2Cu+H]5+	C84H120N27O28Cu2	416.15	416.19	20
	[Aβ16+4H]4+	C84H123N27O28	489.48	489.52	100
	[Aβ16+Cu+2H]4+	C84H121N27O28Cu	504.70	504.77	80
	[Aβ16+2Cu]4+	C84H119N27O28Cu2	519.93	520.04	30
	[Aβ16+3H]3+	C84H122N27O28	652.30	652.44	55
	[Aβ16+Cu+H]3+	C84H120N27O28Cu	672.60	672.78	40
Aβ16–Cu2+–Tol	[T+H]+	C14H21NO5	274.07	274.14	0.2
	[Aβ1-16+5H]5+	C84H124N27O28	391.78	391.76	80
	[Aβ1-16+Cu+3H]5+	C84H122N27O28Cu	403.96	404.03	10
	[Aβ1-16+4H]4+	C84H123N27O28	489.48	489.49	100
	[Aβ16+Cu+2H]4+	C84H121N27O28Cu	504.70	504.76	10
	[Aβ16+2Cu]4+	C84H119N27O28Cu2	519.93	519.99	1
	[Aβ16+3H]3+	C84H122N27O28	652.30	652.43	25
	[Aβ16+Cu+H]3+	C84H120N27O28Cu	672.60	672.77	5

TABLE 3. ESI‐MS characterization of GSH–Tol–Cu2+ mixtures.

Sample	Species	Assignment	Calcd. (m/z)	Exp. (m/z)	RI (%) a
GSH	[2GSH+H]+	C20H35N6O12S2	615.17	615.39	100
GSH–Cu2+	[GSSG+H]+	C20H33N6O12S2	613.16	613.30	100
	[CuGSSG‐H]+	C20H31N6O12S2Cu	674.07	674.37	25
GSH–Tol	[2GSH+H]+	C20H35N6O12S2	615.17	615.38	100
GSH–Tol–Cu2+	[Cu2T−4H+H2O]+	C14H9NO6Cu2	412.90	413.27	60
	[Cu2T−4H+CH3OH]+	C15H11NO6Cu2	426.92	427.35	25
	[T2+H]+	C28H23N21O	547.13	547.46	15
	[GSSG+H]+	C20H32N6O12S2	613.16	613.19	70
	[CuGSSG‐H]+	C20H31N6O12S2Cu	674.07	674.17	35

Full text

1. Introduction

Tolcapone (3,4‐dihydroxy‐4′‐methyl‐5‐nitrobenzophenone, Tol) is a catechol‐O‐methyltransferase (COMT) inhibitor approved as an adjunct to levodopa and carbidopa for the treatment of Parkinson’s disease (PD) patients with motor fluctuations [1]. By inhibiting COMT, Tol increases the levels of dopamine, a neurotransmitter that is diminished in PD [1]. In addition to this role, it was observed that, by enhancing the dopaminergic tone in the prefrontal cortex, Tol improved cognitive control, working memory, and saccadic eye movements, linking the drug neurocognitive modulation with observable changes in eye control functions, showing beneficial effects in neuropsychiatric conditions [2].

Moreover, mounting research suggests that Tol interacts with amyloid proteins implicated in the pathogenesis of Alzheimer’s disease (AD), raising interest in its potential relevance beyond PD and into broader neurodegenerative and neuropsychiatric contexts, including ocular disorders. Indeed, beyond the CNS, Aβ accumulation has been associated with ocular neurodegeneration: in experimental glaucoma, Aβ localizes with apoptotic retinal ganglion cells (RGC), and pharmacological targeting of the Aβ pathway reduces glaucomatous RGC apoptosis; in age‐related macular degeneration (AMD), Aβ has been found within drusen deposits in the retinal pigmented epithelium (RPE), along with metal dishomeostasis, indicating an AD‐like amyloid signature in retinal aging and disease [3]. Previous research has examined the TTR ability of Tol to interact with biologically relevant proteins in neurological disorders, such as both wild‐type (WT‐TTR) and mutant transthyretin (TTR) variants [4], α‐synuclein (αSyn) [5], and amyloid‐beta (Aβ) [6]. Several studies investigated the effects of Tol on Aβ aggregation and toxicity. Tol has been found to exhibit a half‐maximal inhibitory concentration (IC_50_) of approximately 3.2 μM on Aβ_42_ fibrillization [7]. This IC_50_ value is comparable to or even better than several other known Aβ inhibitors such as epigallocatechin‐3‐gallate, curcumin, resveratrol, and other peculiar flavonoids [8–10].

The researchers found that Tol and its derivative Tol‐D, in addition to reducing aggregation, protected against the toxic effects of Aβ in cell and animal models [7, 11]. The current understanding of AD emphasizes the crosslink among different pathogenic elements present in the disease, such as proteinopathy, oxidative stress, and metal ion dyshomeostasis [12]. Proteinopathies refer to neurodegenerative disorders characterized by the misfolding and abnormal accumulation of proteins, particularly aggregation‐prone species [13]. In AD, for instance, aggregated Aβ peptides form extracellular plaques, and Tau protein forms intracellular neurofibrillary tangles—their co‐occurrence and interplay can trigger synaptic dysfunction and cell death [14]. On the other hand, the metal ion hypothesis focuses on the role of metal ion imbalance in the disease process, leading to both loss‐of‐function and gain‐of‐toxicity effects through dysregulation and misplacement of metal ions [15, 16]. Aβ and metal ions contribute to neurodegeneration both independently and synergistically: Zn^2+^ and Cu^2+^ ions, which are found in close proximity to Aβ aggregates in senile plaques, can interact with Aβ peptides and influence their aggregation [17–19]. Furthermore, metal ion dyshomeostasis also leads to oxidative stress as redox‐active metal ions (such as copper) can generate reactive oxygen species (ROS), which can cause cellular damage and contribute to neuronal dysfunction and degeneration [20]. Pioneering studies have further demonstrated that rationally designed small molecules can modulate Cu–Aβ interactions, redirect aggregation pathways, and suppress metal‐induced oxidative stress, thereby validating metal: Aβ species as druggable targets [21–24].

Tol, that fits within this framework, is structurally a nitrocatechol‐functionalized benzophenone, bearing two hydroxyl groups on an aromatic ring. Due to the structural similarity, we can consider Tol as a polyphenol‐like compound (Figure 1) [6, 25]. Polyphenols possess the ability to bind transition metal ions, including copper, and this property has led to their recent proposal as metal‐binding systems in the context of diseases related to metal dyshomeostasis, including AD [26, 27]. A few studies have explored the formation of copper–polyphenol complexes, revealing that hydroxyl groups within the polyphenol structure serve as the primary points of metal anchoring [28].

Chemical structure of tolcapone with the two vicinal hydroxyl groups emphasized in gray. This structural motif closely resembles that of several well‐known natural polyphenols—such as catechin, quercetin, epigallocatechin, apigenin, resveratrol, luteolin, rosmarinic acid, and morin.

However, despite the growing interest in modulating Aβ aggregation, neither the interaction between monomeric Aβ and Tol, nor the effects of Tol on metal‐associated Aβ species have been previously explored. Moreover, the potential for Tol to interact with metals through specific binding sites on its structure has not yet been assessed. This altogether represents a significant gap, particularly considering that in AD brains, such interactions are likely significant due to the abnormal accumulation of both Aβ peptides and metal ions.

To address these points, we employed a series of experimental techniques—including UV–vis spectroscopy, circular dichroism (CD), electrospray ionization mass spectrometry (ESI‐MS), and surface plasmon resonance (SPR)—to characterize the binding properties of Tol not only with Aβ–copper complexes but also with the copper‐free Aβ peptide. Notably, SPR analysis helped to reveal aspects of the binding affinity and kinetics of Tol, shedding light on the unexplored molecular interactions with Aβ. Furthermore, the antioxidant activity of Tol has also been tested in vitro using various assays.

In this study, we demonstrate that Tol directly binds Aβ, both in their copper‐free and copper‐bound forms, and significantly influences their copper‐induced aggregation behavior. Tol, because of its structural and chemical properties, may exert multiple protective effects in the context of AD: acting as an antioxidant, preventing copper binding to Aβ, or even sequestering copper ions already bound to Aβ. This latter activity could potentially restore disrupted copper homeostasis by enabling the intracellular release of copper in a more regulated manner.

2. Materials and Methods

2.1. Materials

Tol was obtained from TCI (Tokyo Chemical Industry Co., Ltd. Boereveldseweg 6—Haven 1063 2070 Zwijndrecht, Belgium) and used directly. Aβ_1-40_ (Aβ) and Aβ_1-16_ (Aβ_16_) peptides were purchased from Bachem (Hauptstrasse 144, 4416 Bubendorf Switzerland). 3‐(N‐Morpholino) propanesulfonic acid (MOPS, ≥ 99.5%), N‐(2‐hydroxyethyl) piperazine‐N′‐(2‐ethanesulfonic acid) sodium salt (HEPES, ≥ 99.5%), ascorbate, and 2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) (ABTS, 98%) were purchased by Merck. Cu^2+^ standard solutions (Cu(ClO_4_)2) were standardized via complexometric titration with EDTA, employing murexide as the indicator.

2.2. UV–Vis and CD Spectroscopy

UV–vis spectra were recorded on a V‐670 JASCO spectrophotometer. CD measurements were performed on a JASCO spectropolarimeter (model J‐1500). CD spectra represent the average of at least five scans. Spectrophotometric titrations were carried out to determine the conditional stability constants of Tol.

2.3. Mass Spectrometry

The ESI‐MS experiments were conducted on a Q‐Tof Ultima API (Waters), a high‐resolution Q‐TOF instrument whose nominal resolving power (approximately 10,000–17,000 FWHM) and typical mass accuracy (a few ppm when properly calibrated) correspond to absolute uncertainties of about 0.002–0.005 Da in the m/z range relevant to our study (approximately 400–1000), as detailed in the Waters Q‐Tof Ultima API user manual. Under these conditions, the third and fourth decimal digits of an m/z value fall within the measurement’s intrinsic error, and, therefore, we report m/z values in the main text and tables with two decimal places. The source parameters were consistently set as follows: cone voltage at 35 V, source block temperature at 70°C, and desolvation temperature at 100°C, with a cone gas flow of 30 L/hr and a desolvation gas flow of 500 L/hr. The acquisition range was set between 50 and 2000 m/z, with scan and interscan times of 1.0 and 0.1 s, respectively.

Tol solutions and Cu^2+^ complexes for ESI‐MS studies were prepared by adding an aqueous Cu^2+^ solution (for copper complex analysis) to an aqueous solution of the ligand, the latter obtained from a 5 mM stock solution in ethanol. The presence of ethanol in the solvent ensures adequate solubility of the drug under the experimental conditions, particularly for the analysis of the copper complexes. The ESI‐MS parameters were adjusted to optimize the signal response.

2.4. SPR Measurements

SPR experiments were carried out on an Octet SF3 system (Sartorius) using CDL sensor chips.

Aβ_1-40_ peptide was immobilized onto two of the three active flow channels via standard amine coupling [29–31]. The peptide was dissolved in 10 mM sodium acetate buffer (pH 4.2) at concentrations of 15 and 30 μM in two different microchannels. Prior to immobilization, the sensor surface was activated with a mixture of 0.4 M N‐ethyl‐N′‐(3‐dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.1 M N‐hydroxysuccinimide (NHS), enabling the formation of reactive succinimide esters. After peptide immobilization, the remaining active esters were blocked with 1 M ethanolamine (pH 8.5). The unbound material was removed by injecting a regeneration solution containing 10 mM NaOH and 1 M NaCl.

The immobilization efficiency was assessed in terms of surface density, yielding an average response of 450 response units (RUs), corresponding to approximately 1.05 pmol/mm^2^ of covalently bound peptide.

Tol solutions were prepared by serial dilution in buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v/v Tween‐20, pH 7.4) supplemented with 5% ethanol to improve solubility and ensure refractive index consistency. Analyte injections were performed in triplicate at a flow rate of 30 μL/min for 2 min. The interaction between Tol and Aβ was performed at 30 μL/min to avoid the mass transport effect. Between cycles, the sensor surface was regenerated by injecting 10 mM glycine/HCl (pH∼2.0) for 30 s at the same flow rate.

Evaluation of sensorgrams was performed using Octet SPR Analysis version 5.0.1 Build 5 2021 Sartorius BioAnalytical Instruments, Inc.

2.5. Turbidity Assay for the Evaluation of the Inhibition of Metal‐Induced Aβ Aggregation

The turbidity assay was performed to evaluate the ability of Tol to inhibit Aβ aggregation induced by Cu^2+^ ions. The assay was conducted in HEPES buffer, using a fixed molar ratio of Aβ:Cu^2+^:Tol of 1:1:2. Samples were incubated at 37°C for 3 h, after which absorbance was measured at 405 nm. Turbidity was assessed by comparing the absorbance of each sample to that of its corresponding control lacking Aβ. To ensure that the absorbance of the Tol–copper complexes does not obscure or interfere with the turbidimetry assays, these were repeated at 600 nm. Each experiment was carried out in triplicate or more. Control samples containing only Aβ and Cu^2+^ were included as positive controls to confirm the occurrence of metal‐induced aggregation in the absence of the ligand.

2.6. AA Oxidation Assay

The ascorbic acid (AA) oxidation assay was conducted according to a previously used protocol [32]. The oxidation of AA was monitored by measuring the UV–vis absorbance at a wavelength of 265 nm, which is related to the unoxidized form of AA. The assay was performed in a water solution of 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES buffer, 50 mM) at pH 7.4. Free Cu^2+^ or Cu^2+^ complexes were added to the AA solution (100 μM) inside a cuvette with a path length of 1 cm. The concentrations of Cu^2+^ and ligand were [Cu^2+^] = 2 μM and [Tol] = 4 μM, respectively. The absorbance and ellipticity of the solution were monitored over a period of 3000 s. Kinetic measurements of AA oxidation were carried out in triplicate using independent solutions prepared daily. To obtain accurate measurements, the absorbance of the Cu complexes at λ = 265 nm under the same conditions was used as a blank, even though its contribution was minimal.

2.7. Trolox Equivalent Antioxidant Capacity (TEAC) Assay

This assay measures the antioxidant capacity (TEAC) of the tested compounds by comparing their ability to scavenge the ABTS^•+^ (2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid)) radical cation relative to 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid (Trolox).

To prepare the ABTS^•+^ radical cation, ABTS (7 mM) was reacted with persulfate (2.45 mM) in water. After 16 h in the dark at 25°C, the resulting solution was diluted in a cuvette with phosphate buffer (5 mM, pH 7.4) to obtain an absorbance of 0.70 ± 0.05 at 734 nm.

The absorbance of ABTS^•+^ was measured spectroscopically for 6 min at 734 nm in the presence of increasing concentrations of the tested samples (ranging from 3 × 10^−6^ to 3 × 10^−5^ M). The decrease in absorbance indicated the reaction between ABTS^•+^ and the tested mixture, with negligible influence from possible side reactions. The TEAC values were calculated at different time points, normalized to Trolox, and reported as means ± standard deviations of three replicates.

3. Results and Discussion

3.1. Determination of Aβ–Tol Binding Affinity

Several recent studies have highlighted the ability of Tol to inhibit both the aggregation and cytotoxicity of Aβ peptides [7]. Despite this growing evidence of functional effects, little is known about the molecular details of the interaction between Tol and Aβ. To gain deeper insights into this interaction, we employed SPR to directly evaluate the binding affinity and kinetics between Aβ and Tol.

SPR directly provides the kinetic parameters of association (ka) and dissociation (kd), from which the equilibrium dissociation constant (K _ D _) can be derived as the ratio kd/ka. This thermodynamic constant quantitatively describes the overall affinity between the ligand and the analyte [31].

From analysis of the sensorgrams (Figure 2), the kinetic parameters of the interaction were determined by applying the 1:1 two‐state binding model, allowing calculation of the corresponding K _ D _. In addition, the K _ D _ value was also obtained from the dose–response plot (Figure S1, SI), in which the maximum SPR RUs from the steady‐state phase were plotted against the respective Tol concentrations (Figure 2).

Sensorgram of the interaction between varying Tol concentrations (47–750 μM) and immobilized Aβ1‐40 on the CDL sensor chip, showing increasing SPR response (RU) with rising analyte concentrations. Data were fitted to a 1:1 two‐state binding model (red lines).

The resulting K _ D _, extrapolated as a weighted average of the individual experimental values and accounting for the uncertainty of each measurement, was 2.20 ± 0.01 mM. This K _ D _ suggests a low affinity of Tol for the monomeric form of the Aβ peptide immobilized on the sensor chip. This finding supports the hypothesis proposed in the previous study by Di Giovanni et al., suggesting that Tol preferentially targets intermediate stages of Aβ aggregation, rather than binding the monomer with high affinity and preventing oligomer formation [6].

3.2. Metal Complexes

Tol belongs to the family of COMT (EC 2.1.1.6) inhibitors developed by Orion Pharma [33]. In the mechanism of action, Tol is supposed to bind the Mg^2+^ ion in the active site of COMT enzyme with its two hydroxyl groups. The binding of copper that is a biologically relevant metal ion, especially in the context of neurodegenerative disorders, was evaluated here for the first time through mass spectrometry and UV–vis spectroscopy.

The positive ion mode ESI‐MS spectrum of a water solution of Tol and Cu^2+^ (in equimolar ratio) is shown in Figure 3. In addition to the pseudo‐molecular ion [M+H]^+^ of Tol at m/z 274.14 and to the peak at m/z 547.14, imputable to its dimer, intense cluster signals indicating the presence of copper are observed. In particular, the peak at m/z 609.09 is assigned to a complex formed by two Tol molecules and one copper atom (Table 1). Cu^2+^ was initially present in solution, but a reduction of Cu^2+^ to Cu^+^ is a common occurrence in the gas phase as often reported in the case of ESI‐MS of Cu complexes [34]. Other peaks are observed at m/z 394.98, 412.98, and 427.00, attributable to dinuclear copper species (Table 1).

ESI‐MS spectrum of Cu2+–Tol 1:1.

The ability of Tol to bind copper ions was also monitored through the titration of Tol with increasing concentrations of Cu(ClO_4_)2 in MOPS (pH 7.4), followed by UV–vis spectral acquisition. The results are presented in Figure 4. In MOPS at physiological‐like conditions, the spectrum of Tol exhibits four main absorbances at 242, 268, 345, and 423 nm resulting from electronic transitions in the benzene rings, in the double C=O bond in the nitro group [35]. The UV–vis spectrum of Tol is strongly influenced by pH (Figure S2, SI). The electronic effect of the NO_2_ group lowers the pKa of Tol as compared to catechol with pK_a1_ values around 4.5 and 10.2 for pK_a2_ [36]. As a result of this, at physiological pH, the monoanionic form of Tol is largely predominant (ca. 99%, Figure S3) and the UV–vis spectrum observed in MOPS is that of the monoanionic form (Figure S2, SI).

UV–vis titration of Cu2+ (0–2 mol equiv) into a solution of Tol at pH 7.4 (0.01 m MOPS/dioxane 70:30). CTol = 4.00 × 10−5 M.

Upon successive additions of Cu^2+^, the band at 348 nm decreases, whereas the bands at 242 nm and 268 significantly change, showing a hyperchromic and bathochromic shift. The analysis of the molar ratio graph at 348 nm suggests the formation of the species CuL_2_, as the curve showed a clear break at the Cu:Tol ratio of approximately 0.5. The conditional logβ values for the Cu^2+^ complexes of Tol were also determined under the same experimental conditions. Precise identification of the Cu^2+^ complex species and their corresponding conditional stability constants was achieved by analyzing multiple titrations and conducting multiwavelength analysis of spectral data. The data analysis converged to these values and species for the CuL (6.2(2)) and CuL_2_ (11.5 (3)) complexes.

3.3. Interaction of Tol With Copper–Aβ

Since AD is characterized by the accumulation of metal ions—particularly Cu^2+^—in senile plaques, and these ions are known to influence both the fibrillation and toxicity of Aβ peptides [37], we examined the ability of Tol to interact with copper–Aβ complexes. Specifically, the direct interactions between Cu^2+^–Aβ_16_ and Tol were assessed using ESI‐MS, UV–vis spectroscopy, and CD.

Aβ_16_ was chosen as a model peptide because it contains the metal‐binding sites located at the N‐terminal region of full‐length Aβ, and its interaction with Cu^2+^ is independent of the overall peptide length. Upon addition of Tol at equimolar concentrations, the UV–vis spectrum of the ternary mixture (Cu^2+^–Aβ_16_–Tol) more closely resembles that of the Cu^2+^–Tol complex prepared in equimolar amounts than that of free Tol, suggesting that Tol competes with Aβ_16_ for Cu^2+^ binding. This observation indicates that Tol is able to coordinate Cu^2+^, displacing it from the peptide. Nonetheless, a fraction of Tol likely remains unbound under these conditions. Analyzing only the UV–vis data, however, the formation of a ternary complex involving Cu^2+^, Aβ_16_, and Tol cannot be definitively ruled out. ESI‐MS analysis does not support the existence of such a species but suggests that Tol effectively competes with Aβ_16_ for Cu^2+^ binding, leading to the extraction of the metal ion from the peptide complex.

As previously reported, the mass spectrum of a 1:1 mixture of Aβ_16_ and Cu^2+^ displays characteristic peaks consistent with metal coordination. Table 2 summarizes the main charged species detected by ESI‐MS in the analysis of Aβ_16_ and its Cu^2+^‐bound complexes. The experimental masses (Exp) are in agreement with the calculated values (Calc.d), confirming the assignment of the observed peaks. The predominant species is the quadruply protonated peptide [Aβ_16_+4H]^4+^, which exhibits the highest relative intensity (RI = 100%), consistent with the high ionization efficiency and stability of this charge state.

Among the Cu^2+^‐containing species, the mononuclear complexes are clearly dominant. For instance, the [Aβ_16_+Cu+2H]^4+^ species (m/z 504.77) shows a high relative intensity (RI = 80%), suggesting the coordination of a single Cu^2+^ ion under the experimental conditions. Similarly, the [Aβ_16_+Cu+3H]^5+^ and [Aβ_16_+Cu+H]^3+^ species are also prominent (RI = 40% each), further supporting the prevalence of the 1:1 Aβ_16_:Cu^2+^ stoichiometry.

Species with 1:2 Aβ_16_:Cu^2+^ stoichiometry are also detected but with significantly lower relative intensities. The [Aβ_16_+2Cu+H]^5+^ and [Aβ_16_+2Cu]^4+^ ions show RI values of 20% and 30%, respectively, indicating that although the peptide can accommodate two Cu^2+^ ions, these complexes are less favored under the tested conditions.

To sum up, the data confirm that Aβ_16_ readily forms stable complexes with one Cu^2+^ ion, with multiple detectable charge states. This pattern provides a clear starting point for evaluating how external ligands (e.g., Tol) influence the metal binding and peptide speciation.

It is noteworthy that the intensities of the peaks of Cu^2+^–Aβ_16_ (m/z 672.77, 519.99, 504.76, and 404.03) dropped significantly when Tol was added. In particular, for the 1:1 Cu^2+^–Aβ_16_ complex, the peak at m/z 404.03, which can be assigned to the [Aβ_16_+Cu+3H]^5+^ species, showed a decrease in relative intensity from approximately 40% to 10%. Similarly, the peak at m/z 504.76, due to the [Aβ_16_+Cu+2H]^4+^ species, dropped from 80% to 10%, and the peak at m/z 672.77, corresponding to the [Aβ_16_+Cu+H]^3+^ species, decreased from 40% to 5%. These results clearly indicate a substantial disruption of the Cu^2+^–Aβ_16_ complex upon the addition of Tol.

The mass spectrometry data are keeping with the results obtained from UV–vis and CD spectroscopy. The significant decrease in the relative intensities of the Cu^2+^–Aβ_16_ complexes observed by ESI‐MS upon Tol addition mirrors the spectral changes detected by UV–vis and CD, which are consistent with copper displacement or redistribution (Figure 5(a)). Overall, all three techniques indicate that Tol effectively competes with Aβ_16_ for Cu^2+^ binding.

FIGURE 5(a) UV–vis spectra of Tol alone (3.6 × 10^-5^ M), Tol–Cu^2+^ (1:1), and Cu–Aβ_16_–Tol (1:1:1) in MOPS (2 mM, pH 7.4); (b) turbidity measurements of Aβ (alone, black bar) solutions incubated with Cu^2+^ ions in the absence (white bar) or in the presence of Tol (red bar) and EDTA (blue bar) and tacrine (fuchsia bar) as positive and negative controls. Each ΔA_405_ value represents the mean of three independent experiments, and error bars indicate standard deviations.(a)(b)

This finding is particularly notable when considered alongside the literature on Cu(II)–Aβ affinity. Calorimetric and competition studies at pH 7.4 have shown that the N‐terminal fragment Aβ_1-16_ binds Cu(II) with conditional dissociation constants in the subnanomolar range (Kd ≈ 10^−10^–10^−9^ M) [38]. For full‐length Aβ_1-40_ and Aβ_1-42_, Cu(II) binding is reported to be of comparable or slightly higher affinity, with a consensus Kd window of ∼50–100 pM for both monomeric and fibrillar peptides [39, 40]. Our UV–vis, CD, and ESI‐MS data consistently demonstrate that Tol is still capable of partially displacing Cu(II) from Aβ. This indicates that Tol can alter Cu–Aβ speciation under physiological‐like conditions, leading to a partial release of Cu^2+^ from the Aβ_16_ binding sites.

The ability of the compound Tol to inhibit Cu^2+^‐mediated aggregation of Aβ was assessed using a turbidity assay, an established method for evaluating the formation of both fibrillar and amorphous Aβ aggregates. Turbidity measurements were performed on equimolar solutions of Aβ and Cu^2+^, in the presence or absence of Tol. For comparison, EDTA was used as a positive control due to its well‐known Cu^2+^‐chelating properties and the ability to prevent metal‐induced protein aggregation. Tacrine, a clinically used AChE inhibitor that does not chelate Cu^2+^, was included as a negative control. As expected, Cu^2+^ significantly promoted Aβ aggregation, as evidenced by the increase in absorbance. The addition of EDTA substantially reduced aggregation, confirming its inhibitory role through copper chelation. In contrast, tacrine did not affect aggregation levels. The presence of Tol led to a marked reduction in Cu^2+^‐induced aggregation, though less pronounced, compared to EDTA. The observed inhibitory effect is in a good agreement with a lower binding affinity of Tol for Cu^2+^ compared to EDTA and is consistent with UV–vis and ESI‐MS data, which indicate that Tol competes with Aβ_16_ for Cu^2+^ coordination and is able to partially extract the metal from the peptide complex. These findings suggest that Tol can modulate Cu^2+^‐induced Aβ aggregation through a metal‐chelation mechanism, albeit with moderate efficacy compared to a stronger chelator such as EDTA. This chelation process leads to the formation of Cu^2+^‐free Aβ species, providing a possible mechanism for the observed inhibition of metal‐induced Aβ aggregation.

3.4. Interaction With Glutathione

Once the extraction of copper from Aβ peptides by Tol in vitro was established, we also investigated if Tol could release copper ions in an intracellular‐like environment. Given that the latter is highly reducing and rich in glutathione (GSH), it is critical to assess whether GSH can interfere with the stability of the copper–Tol complex. To address this, we studied the interaction between GSH, copper, and Tol, aiming to simulate the redox environment encountered in cell. This investigation was carried out using ESI‐MS, UV–vis, and CD, providing complementary insights into the possible displacement of copper from Tol by GSH. UV–vis spectroscopy was employed to monitor the interaction between the Cu^2+^–Tol complex and GSH (Figure S4, SI). Introducing one equivalent of GSH to the Cu^2+^–Tol solution in MOPS buffer (20 mM, pH 7.4) under aerobic conditions (atmospheric oxygen) caused an immediate spectral shift: a marked decrease in absorbance at 282 nm and a concurrent rise at 347 nm. These changes are consistent with partial dissociation of the complex and the liberation of the free ligand. Upon addition of a second equivalent of GSH, the transformation was complete, indicating full release of copper from the complex. Notably, the final UV–vis spectrum of the 1:1:2 Tol–Cu^2+^–GSH mixture was superimposable to that of free Tol at the same concentration, confirming that the ligand was restored to its unbound form.

Mass spectrometry analyses were also carried out on the mixture 1:1:1 Tol–Cu^2+^–GSH. For comparison, the mass spectra of GSH alone, GSH in the presence of Cu^2+^, and Tol in the presence of GSH were also recorded to enable the identification of key species in the ternary mixture (Table 3). The ESI‐MS spectrum of GSH alone clearly displayed the reduced form of the tripeptide, with a prominent base peak at m/z 615.39 corresponding to the [2GSH+H]^+^ dimeric ion. This peak remained evident in the presence of Tol, indicating that GSH persists in its reduced form. Importantly, no signals corresponding to oxidized glutathione (GSSG) were detected in these samples. When analyzing the spectrum of the ternary mixture Tol–Cu^2+^–GSH, some peaks can be attributed to the presence of Tol alone (see the dimeric adducts at m/z 547.46); other peaks correspond to complex species involving Tol, while glutathione appears only in its oxidized form, either free or complexed with copper at m/z 613.19 and m/z 674.17, respectively. The results indicate that, under intracellular‐like conditions, the complex releases copper upon reaction with excess GSH. This process involves the oxidation of GSH and the concomitant reduction of the copper ion, leading to the dissociation of Tol. The released copper is subsequently sequestered by glutathione (GSH or GSSG) or potentially by biological molecules such as metallothioneins or other copper‐binding proteins. These observations support the hypothesis that Tol may act as a copper chelator in the extracellular environment, specifically in regions characterized by amyloid plaque deposition, where dysregulated copper homeostasis is observed [41]. By binding copper extracellularly, Tol could limit metal‐catalyzed oxidative processes associated with amyloid aggregation and neurotoxicity. Upon cellular uptake and exposure to intracellular reducing agents such as GSH, the complex may undergo redox‐mediated dissociation, leading to the release of copper ions. These released ions could then be sequestered by intracellular metal‐binding proteins such as metallothioneins, thereby contributing to the restoration of cellular metal homeostasis [42].

3.5. Antioxidant Activity

A broad debate is currently ongoing within the scientific community regarding the initial event that triggers the pathogenic cascade of AD [43]. On one side, oxidative stress is proposed as the primary initiating factor; on the other, this role is attributed to Aβ deposition or tau hyperphosphorylation [44–46]. Based on recent experimental findings, several research groups have suggested that oxidative stress represents an early event capable of promoting both the formation of oligomeric Aβ and the dimerization and hyperphosphorylation of tau protein, as well as triggering other pathogenic pathways [47]. In this context, the oxidative stress hypothesis highlights the urgency of developing targeted therapeutic strategies that include the use of antioxidant compounds.

The ABTS radical assay was used to assess the antioxidant activity of Tol in the presence and absence of metals. To quantify radicals that can be scavenged by antioxidants, the TEAC assay is commonly utilized [48]. This assay is based on the ability to scavenge the 2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cation (ABTS˙^+^), converting it into a colorless product. The extent of decoloration caused by antioxidant compounds was measured using UV–vis spectroscopy and compared to Trolox, a well‐known antioxidant, to obtain the TEAC value.

The TEAC values of Tol samples are reported in Figure 6, providing insights into its antioxidant capacity. Additionally, TEAC values of GSH were determined in the same conditions for comparison purposes. GSH, classified as a thiol‐type antioxidant, plays a pivotal role in safeguarding cells against oxidative stress by engaging with the electrophilic components of ROS, serving as a primary and fundamental element of the body antioxidant defense mechanism. Thiols generally undergo oxidation involving a single electron, leading to the creation of thiyl radicals (R–S^•^) by either losing a hydrogen atom from the –SH group or losing an electron from the sulfur. In physiological pH conditions, these thiyl radicals are unstable and may combine again to produce the corresponding disulfide [49, 50].

TEAC values at 1, 3, and 6 min for Tol in MOPS (5 mM, pH 7.4). TEAC values are the average of four independent trials, and error bars show standard deviations.

At pH 7.4, Tol scavenges ABTS^•+^ with comparable rates to those polyphenols. The hydroxyl group on aromatic rings, known for its high reactivity and hydrogen‐donating capability, can be responsible for this free radical scavenging activity. However, the presence of Cu^2+^ decreased the scavenging rate for Tol, as seen from the TEAC value presented in Figure 6, while the Cu^2+^ ion did not interact with ABTS^•+^. CuTol_2_ species contains two ligands, consequently diminishing the active concentration of Tol as a radical scavenger when in equilibrium with the complex.

3.6. Ascorbate Assay

The oxidation of ascorbate is a widely used assay to evaluate the redox activity and ROS‐generating potential of metal complexes [51–53]. In particular, Cu–Aβ complexes have been shown to catalyze the production of ROS species including hydroxyl radicals (^•^OH), contributing to oxidative stress in AD [39, 54]. Monitoring the kinetics of ascorbate oxidation by UV–vis spectroscopy provides a sensitive and easy method to evaluate the catalytic activity of redox‐active complexes. This approach allows us to indirectly estimate ROS production and offers insight into redox behavior of the metal center. The UV spectrum of AA at pH 7.4 displayed a prominent absorption peak at 265 nm (with a corresponding CD band at 250 nm). Upon oxidation, this peak disappeared, allowing the process to be monitored by measuring absorbance at 265 nm. From experiments performed in the presence of AA, it is observed that Tol alone does not significantly cause the oxidation of ascorbate. When copper is present, this oxidation occurs, as evidenced by the decrease in the UV and CD band due to reduced ascorbate at 250 nm, and the presence of Tol inhibits the oxidation process, especially when Tol is present at a concentration twice that of copper (Figure 7). The interpretation of these results could be useful for the understanding of the chemical behavior of Tol in a biological environment, especially in the presence of transition metals such as copper. These metals can be involved in significant redox reactions in biological systems. The fact that Tol inhibits the oxidation of AA in the presence of copper might suggest that Tol interferes with certain metal‐catalyzed redox processes or rather the copper–Tol complex does not have the same ability as copper alone to catalyze the oxidation of AA. This information could be relevant for its use as a drug. Overall, this assay suggests that Tol has a protective effect against copper‐induced oxidation of AA.

Representative kinetics of AA consumption, measured by UV–vis at 265 nm, of Cu2+ alone, Tol alone, and Cu2+ complexes of Tol. Conditions: AA 100 μM, ligand 4.0 μM, Cu2+ 2.0 μM, HEPES 50 mM pH 7.4, 25°C, cuvette path length of 1 cm.

4. Conclusions

This study gives insights into the multifaceted interactions between Tol and Aβ, highlighting its potential role in modulating both copper‐free and copper‐bound Aβ species. In particular, our data demonstrate that Tol directly binds to Aβ monomer with an affinity in the millimolar range, as determined by SPR analysis. Spectroscopic and mass evidence support its capacity to interfere with copper–Aβ interactions, either by competing for metal binding or by destabilizing preformed Aβ–Cu complexes. In addition, the observed radical scavenging activity of Tol reinforces its potential as a protective agent against oxidative stress commonly associated with Aβ–copper toxicity. Hence, Tol may contribute to the disruption of pathological Aβ aggregation pathways through both metal chelation and direct interactions with Aβ species. These results open promising avenues for the development of Tol derivatives as therapeutic tools in the treatment of neurodegenerative disorders that affect both the CNS and the ocular compartment.

Funding

The authors acknowledge the Ministry of Health and Fondazione Roma for their support. This research was supported by MUR: PRIN 2022‐P2022AW2H9 “Molecular details on the early phase of amyloid beta peptides aggregation: a multilevel approach based on carbon dots fluorescence and diffusion coefficients measurements to unveil the pathogenic molecular mechanisms at the base of Alzheimer’s disease”; PRIN2022 project (n. 2022BTMYWZ; CUP code: B53D23015260006) entitled “New mEtal‐baSed agenTs against Orphan tumoRs–NESTOR” [announcement D.D. 104 del 02/02/2022 ‐ PNRR per la Missione 4, Componente 2, Investimento 1.1], financed by the European Union—NextGenerationEU.”

This work was also funded by HORIZON‐EIC‐2024‐PATHFINDEROPEN‐01 under grant agreement Project N. 101185661 ‐‐ ERMES and the Italian Ministry of Health: Piano di Sviluppo e Coesione del Ministero della Salute 2014–2020, Project: Pharma‐HUB ‐ Hub per il riposizionamento di farmaci nelle malattie rare del sistema nervoso in età pediatrica (CUP​ E63C22001680001 ‐ ID T4‐AN‐04). VO also thanks Piaceri2024‐TRACE. Open access publishing facilitated by Universita degli Studi di Catania, as part of the Wiley ‐ CRUI‐CARE agreement.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting Information

Supporting information associated with this article includes:

Figure S1. Dose–response plot: equilibrium SPR responses (RU) at the sensorgram plateau (100–120s) were plotted against Tol concentration. The line denotes the nonlinear fit used to calculate the equilibrium dissociation constant (K D).

Figure S2. UV‐vis of Tol at different pHs.

Figure S3. UV–vis spectra of Tol alone and the mixtures Tol/Cu^2+^ 1:1, Tol/Cu^2+^/GSH 1:1:1, and Tol/Cu^2+^/GSH 1:1:2 at pH 7.4 (0.01 M, MOPS).

Figure S4. Distribution diagram of acid–base species of Tol (AH^2^ represents the totally protonated species of Tol) as a function of pH.

Figure S5. Turbidity measurements of Aβ (alone, black bar) solutions incubated with Cu^2+^ ions in the absence (white bar) or in the presence of Tol (red bar) and EDTA (blue bar). Each ΔA^600^ value represents the mean of three independent experiments, and error bars indicate standard deviations.

Supporting information

Supporting Information Additional supporting information can be found online in the Supporting Information section.