Multi-target-directed therapeutic strategies for Alzheimer's disease: controlling amyloid-β aggregation, metal ion homeostasis, and enzyme inhibition

Abstract

Alzheimer's disease (AD) is the most prevalent neurodegenerative dementia, marked by progressive cognitive decline and memory impairment. Despite advances in therapeutic research, single-target-directed treatments often fall short in addressing the complex, multifactorial nature of AD. This arises from various pathological features, including amyloid-β (Aβ) aggregate deposition, metal ion dysregulation, oxidative stress, impaired neurotransmission, neuroinflammation, mitochondrial dysfunction, and neuronal cell death. This review illustrates their interrelationships, with a particular emphasis on the interplay among Aβ, metal ions, and AD-related enzymes, such as β-site amyloid precursor protein cleaving enzyme 1 (BACE1), matrix metalloproteinase 9 (MMP9), lysyl oxidase-like 2 (LOXL2), acetylcholinesterase (AChE), and monoamine oxidase B (MAOB). We further underscore the potential of therapeutic strategies that simultaneously inhibit Aβ aggregation and address other pathogenic mechanisms. These approaches offer a more comprehensive and effective method for combating AD, overcoming the limitations of conventional therapies.

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1. Introduction

Among neurodegenerative diseases, Alzheimer's disease (AD) is the most prevalent form of dementia, characterized by a progressive decline in cognitive function and memory.¹ Despite extensive research since its discovery in 1906, effective treatments for this fatal disorder continue to be elusive.² The pathophysiology of AD remains incompletely understood, highlighting the challenges in identifying its underlying causes and their interconnections.³ Pathological features found in AD-affected brains include amyloid-β (Aβ) aggregate deposition, metal ion dysregulation, oxidative stress, impaired neurotransmission, neuroinflammation, mitochondrial dysfunction, and neuronal cell death, as illustrated in Fig. 1a.^4–15 While no single hypothesis can fully explain the complex pathology of AD, it is evident that multiple factors simultaneously contribute to its progression.¹⁶

Fig. 1 Pathological features in AD and design strategies for multi-target-directed chemical reagents. (a) Multifaceted pathology associated with AD. (b) Schematic illustration of design strategies (linkage, fusion, and incorporation) to develop small molecules against Aβ aggregation, metal ion dyshomeostasis, and enzyme dysfunction simultaneously.

Several U.S. Food and Drug Administration (FDA)-approved or experimental single-target-directed therapies, ranging from small molecules and monoclonal antibodies aimed at disrupting Aβ aggregation^17–21 to metal chelators^22–27 and neurotransmission enhancers,^28–33 have not demonstrated significant disease-modifying effects.^3,16,34 Although these treatments effectively modulate specific pathological mechanisms, the multifactorial characteristic of AD likely limits their overall efficacy and can lead to severe side effects. Considering the complexity of AD, it is crucial to prioritize the development of multi-target-directed strategies that engage the multifaceted nature of the disease in drug design and discovery.^3,35,36 Given recent advancements in targeting Aβ as a clinical approach and its direct interactions with factors like metal ions and enzymes under pathological conditions, this review primarily illustrates Aβ, metal ions, and AD-related enzymes as pathogenic elements (Fig. 1a). The objective is to provide an overview of integrated therapeutic designs while evaluating the limitations of traditional single-target-directed treatments.^17–21 Additionally, this review explores frameworks that simultaneously address these features, focusing on multi-target-directed ligands (MTDLs) and discussing their potential advantages in treating AD, which highlights promising directions for future therapeutic development (Fig. 1b).

2. Single-target-directed drugs and their limitations

2.1 Aβ peptides

In 1984, Aβ peptides were identified as the central component of extracellular amyloid plaques found in the brains of patients.^1,37 Since then, they have been recognized as a critical driver of AD pathology, leading to the development of the “amyloid cascade hypothesis,” which remains a prominent theory in understanding AD pathogenesis.^1,38 As illustrated in Fig. 2a, Aβ peptides are derived from a transmembrane protein known as amyloid precursor protein (APP).² APP undergoes processing via non-amyloidogenic and amyloidogenic pathways.^44,45 In the amyloidogenic pathway, sequential proteolytic cleavages by β- and γ-secretases generate intrinsically disordered, aggregation-prone Aβ peptides.^2,46 Due to the imprecise nature of these cleavages, Aβ peptides are yielded in various forms, such as Aβ_1−x, Aβ_4−x, Aβ_8−x, and Aβ_9−x, with Aβ₄₀ and Aβ₄₂ being the most prevalent species found in amyloid plaques (Fig. 2b).⁴⁶

Fig. 2 Recent drug development against Aβ species or BACE1. (a) Illustration of the proteolytic cleavage of APP. Non-amyloidogenic and amyloidogenic pathways occur depending on the combinational action of α-, β-, and γ-secretases. (b) Amino acid sequences of Aβ₄₀ and Aβ₄₂, with side views of 3D reconstructed Aβ fibrils, and top views of their cross-sections from cryo-EM (for Aβ₄₀, PDB 6SHS³⁹; for Aβ₄₂, PDB 5OQV⁴⁰). Amino acid residues responsible for the self-recognition sites are highlighted in bold and underlined, while those forming salt bridges in fibrillar structures are indicated in red and depicted as sticks. Core structures of Aβ fibrils are also shown in stick representation. Reproduced with permission from ref. 39 and 40. Copyright© 2019 Springer Nature and 2017 The American Association for the Advancement of Science. (c) Schematic description of Aβ aggregation and Aβ-lowering monoclonal antibodies. (d) Relative safety and efficacy of Aβ-lowering monoclonal antibodies in patients with sporadic early-symptomatic AD. Monoclonal antibodies approved by the U.S. FDA for AD are shown in red dots, while other monoclonal antibodies are presented in black. Reproduced with permission from ref. 41. Copyright© 2023 Springer Nature. (e) Structure of BACE1 (PDB 1W50 ⁴²), zoom-in view of the target site, and BACE1 inhibitors that have reached phase III clinical trials. The catalytic dyad is presented in red and depicted as sticks. (f) Structure of γ-secretase (PDB 5A63 ⁴³), illustration of the binding pocket, and γ-secretase inhibitors that have progressed to clinical trials. The catalytic dyad is indicated in red with sticks.

Under physiological conditions, the levels of Aβ peptides are tightly regulated.⁴⁷ In the brains of AD patients, however, an imbalance between the production and clearance of Aβ leads to its accumulation.⁴⁷ Fibrils and plaques are believed to form through intermolecular interactions at the self-recognition and hydrophobic C-terminal regions.^39,40 Recent advances in cryogenic electron microscopy (cryo-EM) have elucidated the structures of Aβ fibrils found in the brains of AD patients (Fig. 2b).^39,40 Both Aβ₄₀ and Aβ₄₂ fibrils consist of two stacks of peptides, each constructing a right-handed twist along a central axis. In Aβ₄₀ fibrils, the peptide stack typically contains four sets of cross-β sheets spanning residues A2–S8, Y10–H13, Q15–F19, and I32–L34.³⁹ In Aβ₄₂ fibrils, additional interactions, such as salt bridges between D1 and K28, D7 and R5, and E11 with H6 and H13, are observed, with those involving E11 stabilizing a kink in the N-terminal part of the β-sheet around Y10.⁴⁰ These interactions promote the assembly of monomers into oligomers, protofibrils, and fibrils. Notably, Aβ oligomers are neurotoxic and contribute to cellular dysfunction, inflammation, and synaptic impairment, all of which drive neurodegeneration in AD.⁴⁸

Given that the accumulation of both soluble and insoluble Aβ aggregates can trigger pathological processes in AD,¹⁷ previous research has focused on reducing cerebral Aβ,¹ or diminishing its production to alleviate the Aβ burden.^49,50 These approaches have been pursued either through the use of anti-amyloid monoclonal antibodies^20,41,51 or by inhibiting β- or γ-secretases,⁴⁹ as illustrated in Fig. 2c–f. Recently, the U.S. FDA has approved three human monoclonal antibodies directed against Aβ (Fig. 2c and d): aducanumab (approved in 2021), lecanemab (approved in 2023), and donanemab (approved in 2024). Each of these therapies has shown potential to slow the progression of AD by targeting specific forms of Aβ: aducanumab addresses soluble oligomers and fibrils;²¹lecanemab engages with soluble protofibrils;^17,18donanemab is designed to target amyloid plaques.^19,20 These therapeutics have demonstrated promise in decreasing amyloid plaque burden and potentially delaying cognitive decline.^17–21 Despite these encouraging results, the outcomes of clinical trials have often failed to meet expectations.^18,20,21,52 Confronting Aβ aggregates with anti-amyloid monoclonal antibodies remains controversial due to mixed treatment outcomes and concerns about adverse effects, such as brain swelling and microhemorrhages, collectively known as amyloid-related imaging abnormalities (ARIA).^18,20,21

In recent decades, the inhibition of β-secretases has garnered attention as a therapeutic strategy for AD, with a particular focus on targeting β-site APP cleaving enzyme 1 (BACE1) to reduce the production of Aβ.⁵³ The structure of BACE1, as illustrated in Fig. 2e, includes a protease domain with two catalytic aspartates (D32 and D228) and several sub-pockets within the catalytic clefts (e.g., S1, S1′, S2′, and S3 pockets).^42,54 Amidine-based BACE1 inhibitors have been extensively studied, with some advancing to phase II or III clinical trials. These inhibitors interact with the aspartate dyad through salt bridges and specifically bind to the enzyme's active site.⁵⁴ Notably, verubecestat (MK-8931) and lanabecestat (AZD3293) (shown in Fig. 2e) significantly reduced the levels of Aβ₄₀, Aβ₄₂, and soluble APPβ (sAPPβ) in cerebrospinal fluid (CSF) by up to 80%, accompanied by a modest decrease in plaque load as confirmed by amyloid positron emission tomography (PET) imaging.^55,56 Over fourteen β-secretase inhibitors have been developed, showing promising results in lowering Aβ burden in advanced clinical trials. They were discontinued, however, due to adverse effects, including liver toxicity, cognitive worsening, weight loss, sleep disturbances, and skin rashes.⁵²

The inactivation of γ-secretase has also been investigated to control Aβ production and attenuate the progression of AD.^43,57,58 As depicted in Fig. 2f, γ-secretase shares a similar structure to that of BACE1, containing two catalytic aspartates (D257 and D385) and five cavities (S1, S1′, S2, S2′, and S3′ pockets) within the active site.^43,57,58 Several inhibitors, such as semagacestat (LY450139) and avagacestat (BMS-708163) (Fig. 2f), have been developed to interact with these sites as potential AD treatments.^59,60Semagacestat, was the first γ-secretase inhibitor to enter phase III clinical trials, but it was withdrawn due to the lack of cognitive improvement and the emergence of side effects, including further cognitive decline.^55,56,59 Additionally, some γ-secretase inhibitors exhibited toxic effects, such as an increased risk of skin cancer, likely resulted from off-target interactions with proteins like the Notch receptor, which is involved in cell proliferation via nuclear signaling.⁵⁹ Although recent drug approvals targeting Aβ aggregates highlight their relevance as a biomarker, these challenges underscore the limitations of focusing solely on a single pathological factor, such as Aβ species or its associated secretases, to reverse cognitive impairment. The complexity of AD pathogenesis and the unintended effects of these inhibitors emphasize the need for therapeutic strategies that address multiple pathological factors simultaneously.⁴¹

2.2 Metal ion dyshomeostasis

Metal ions, such as Fe(II/III), Cu(I/II), and Zn(II), play vital roles in physiological processes, including signal transmission and catalytic reactions, through their coordination to various biological Lewis bases.^61–65 These metal ions are distributed between two distinct pools: the static and labile pools. The static pool consists of tightly bound metal ions embedded within proteins, often stabilizing protein structures or occupying catalytic sites of enzymes.^61–66 In contrast, the labile pool contains loosely-bound or surface-exposed metal ions that participate in more dynamic processes, such as signal transmission and regulation.^61–66

The delicate balance between static and labile metal ion pools can be disrupted under pathological conditions, leading to metal ion dyshomeostasis in the brain. In AD-affected regions, such as the hippocampus and amygdala, an increase in unregulated labile copper ions in serum and plasma has been observed.^67,68 Additionally, the concentration of Zn(II) in these areas nearly doubles compared to healthy subjects.⁶⁷ The miscompartmentation of metal ions not only impairs their physiological functions but also induces toxicity, supporting the “metal ion hypothesis” in AD.⁶⁹ Specifically, the mislocalization of metal ions undermines antioxidant systems against reactive oxygen species (ROS), leading to elevated oxidative stress and severe cytotoxicity.⁶⁶ Excess ROS can damage biological components, such as lipids and proteins, and induce neuronal death, which further leads to neurodegeneration.⁶⁹

Extensive research on the regulation of Cu(I/II) and Zn(II) dyshomeostasis has led to the development of several chelators, including trientine, tetrathiomolybdate, dipicolinic acid, ditiocarb, clioquinol, and PBT2, as illustrated in Fig. 3a.^22–27 These chelators are designed to remove excess metal ions concentrated in plaques, prevent redox reactions, and potentially offer therapeutic benefits for AD.^{22–25,72–76} In addition, clioquinol and an 8-hydroxyquinoline derivative, PBT2, were considered ionophores to restore metal ion homeostasis by coordinating metal ions through their nitrogen and oxygen donor atoms, reintroducing them into physiological systems and preventing their pathological interactions with Aβ.^26,27 Despite their initial promise, chelation-based therapies have not been successful in clinical trials, with no significant improvement and severe side effects.^25,77–79 The failure of these therapies may be attributed to several factors, including (i) non-specific binding to target metal ions, (ii) retinal and ocular toxicity with unclear mechanisms, and (iii) an inaccurate understanding of metal concentrations in AD.^25,77–79

Fig. 3 Metal-related pathological factors associated with AD and therapeutic interventions against these components. (a) Examples of metal chelators. (b) Representative coordination modes of Cu(I/II), Zn(II), and Fe(II) to Aβ with a chemical structure of metal-bound L2-b. L2-b, N¹,N¹-dimethyl-N⁴-(pyridin-2-ylmethyl)benzene-1,4-diamine. (c) Structures of MMP9 (PDB 1L6J⁷⁰) and its inhibitor (prinomastat). The Zn(II)-coordination residues are highlighted in red and depicted as sticks. (d) Structures of LOXL2 (PDB 5ZE3 ⁷¹) and its inhibitor (PXS-5505). The Cu(II)-coordination residues and catalytic lysine are indicated in red with sticks. Note that Cu(II) occupies the Zn(II)-binding site in human LOXL2, contrary to the crystallographic illustration.

To address these challenges, recent strategies have focused on regulating various pathological features associated with metal ion dyshomeostasis. Excess metal ions can coordinate to amyloidogenic peptides (e.g., Aβ, illustrated in Fig. 3b), accelerating their aggregation and exhibiting cytotoxic effects.^69,80,81 The formation of metal–Aβ complexes at the synaptic cleft is well-documented, with metal concentrations in Aβ plaques reaching up to 0.4 mM for copper and 1.0 mM for zinc.⁸² Specifically, two major Cu(II)-binding modes to Aβ have been identified depending on pH, as shown in Fig. 3b. At pH ≤ 6.5 (Component I), Cu(II) primarily adopts a 3N1O coordination mode involving the N-terminal primary amine, the backbone carbonyl group between D1 and A2, and two imidazole rings from H6 and H13 or H14.^83–85 Additional oxygen donor atoms from water molecules or carboxylate groups from D1, E3, D7, or E11 are also proposed.^83–85 In contrast, at pH ≥ 8 (Component II), Cu(II) maintains a 3N1O coordination but with different donor atoms: the N-terminal primary amine, a deprotonated amide backbone between D1 and A2, an imidazole ring from H6, H13, or H14, and an oxygen donor atom from the backbone carbonyl group between A2 and E3.^69,83,84,86 Cu(I) binds to Aβ in a linear mode, primarily entailing the imidazole rings from H6, H13, or H14, with a preference for engaging H13 and H14.^87,88 Moreover, Zn(II) coordinates to Aβ through a 2N2O coordination comprising two oxygen donor atoms from the carboxylate groups of D1, E3, or D7 and E11 and two nitrogen donor atoms from the imidazole rings of H6 and H13 or H14 (Fig. 3b).^69,89 Furthermore, although relatively less established, Fe(II) can associate with Aβ through a 3N3O coordination mode. This incorporates three nitrogen donor atoms from the N-terminal primary amine and two imidazoles from H6 and H13 or H14 and three oxygen donor atoms from the backbone carbonyl groups between D1 and A2, between H6 and D7, and the carboxylate group of D1 (Fig. 3b).^90,91

These interactions facilitate Aβ aggregation and elevate oxidative stress by either disrupting ROS scavenging systems or overproducing ROS through Fenton chemistry or Fenton-like reactions in the case of redox-active metal ions. For example, the varying coordination modes of Cu(I) and Cu(II), coupled with the conformational flexibility of native peptides, enable redox reactions at the metal center (E^o′ = 0.28 V vs. NHE⁹²), generating ROS.^90,93 Note that Fe(II/III) unbound and bound to Aβ is also involved in ROS generation; however, the E^o value could not be accurately determined in aqueous media because of Fe(OH)₃ sedimentation.^90,91 Considering the impact of metal ions on Aβ aggregation and ROS formation, targeting metal–Aβ complexes, therefore, appears to be a promising therapeutic strategy compared to controlling individual factors.^94–96 Lim and coworkers rationally designed a small molecule, L2-b (shown in Fig. 3b), to specifically regulate the reactivities of metal–Aβ complexes.^94,97L2-b inhibited metal-induced Aβ aggregation and reduced ROS produced by Cu(I/II)–Aβ complexes.⁹⁴In vivo studies further demonstrated that L2-b treatment mitigated amyloid pathology and improved cognitive deficits in the 5xFAD mouse model, underscoring the importance of confronting and regulating metal–Aβ interactions.⁹⁴ Despite the significance of these findings, no clinical trials addressing metal–Aβ species have been reported to date, to the best of our knowledge.

Dysregulated and miscompartmentalized metal ions can significantly impact the structures and functions of metalloenzymes.^98–100 Among various metalloenzymes, matrix metalloproteinase 9 (MMP9) and lysyl oxidase-like 2 (LOXL2) have emerged as major pathological factors (depicted in Fig. 3c and d), as they are activated by excess labile zinc or copper ions under pathological conditions.^98–105 MMP9, which plays a crucial role in physiological processes, such as embryonic development, angiogenesis, and cell migration, was initially studied for its proteolytic activity against Aβ peptides.^103,104

Research in AD mouse models has shown that MMP9 is overexpressed, contributing to neurotoxicity in hippocampal neurons, cognitive deficits, and the degradation of nerve growth factors, thereby implicating MMP9 activation in the progression of AD.^104,106,107In vivo studies further suggested that suppressing MMP9 can alleviate neurobehavioral deficits, making it a potential therapeutic target.^104,106,107 Current MMP9 inhibitors are designed to bind to its binuclear Zn(II) centers (Fig. 3c). The two Zn(II) ions are coordinated through two main modes: (i) 3N coordination with the imidazole rings of H175, H190, and H205 and (ii) 3N1S coordination with the free thiol of C99 and the imidazole rings from H401, H405, and H411.⁷⁰ The 3N1S coordination serves as the catalytic center, which becomes activated when the free thiol of C99 dissociates, creating a vacant site. Chelator-based therapeutics, such as prinomastat (shown in Fig. 3c), are being explored for their ability to address this vacant Zn(II)-coordination site and competitively inhibit the active site of MMP9.^104,108

In parallel, LOXL2 is another metalloenzyme that contains a catalytic copper center (Fig. 3d).^71,109 The LOX family is responsible for the oxidative deamination of ε-amino groups in lysine residues, converting them to hydroxylysine within collagen and elastin chains. This chemical transformation initiates covalent cross-linking in the extracellular matrix, which is crucial for maintaining the tensile strength and elasticity of connective tissues.^105,109 The redox-active Cu(I/II) in the LOXL2 active site facilitates this process through the 3N1O coordination with a distorted tetrahedral geometry involving three imidazole rings from H626, H628, and H630 and an additional oxygen donor atom from Y689 (Fig. 3d).⁷¹ The catalytic copper then oxidizes the lysine tyrosylquinone (LTQ) cofactor, enabling further deamination of lysines.¹¹⁰

Given the diverse physiological roles of LOXL2, its dysregulation can impact various pathophysiological conditions, such as AD and other vascular diseases.^105,114 For example, LOXL2 activation can induce arterial stiffness, leading to brain microvascular damage and Aβ deposition.¹⁰⁵ Additionally, suppression of LOXL2 has been shown to enhance the clearance of Aβ aggregates, making it an intriguing therapeutic target.¹⁰⁵ LOXL2 inhibitors typically modify the chemical structure of its substrate, the LTQ cofactor, or disrupt the Cu(II)-binding site to halt enzymatic activity.^109,110 Fluoroallylamine-based inhibitors, such as PXS-5505 (shown in Fig. 3d), form a covalent bond with the LTQ cofactor, resulting in irreversible termination of its function.¹¹⁰ Other compounds, such as guaiacol derivatives, have also demonstrated modulatory effects against LOXL2 by binding to the catalytic Cu(II) center and showed potential as AD therapeutics.¹¹⁵ Collectively, metal ion dyshomeostasis drives to inappropriate interactions with Aβ and the activation of metalloenzymes, thereby increasing cytotoxicity. These findings suggest that developing multipotent chemical reagents capable of confronting and controlling pathogenic features associated with metal ion dyshomeostasis is a promising therapeutic strategy for AD.¹¹⁶

2.3 Neurotransmitters and related enzymatic systems

Imbalances in neurotransmission caused by the dysregulation of enzymatic systems represent another pathological factor for AD.^{28,44,103,117–121} Patients with AD often experience diminished signal transmission mediated by neurotransmitters, such as acetylcholine (ACh), dopamine, and serotonin. These neurotransmitters are regulated and degraded by enzymes, including acetylcholinesterase (AChE), monoamine oxidase (MAO), and catechol-O-methyltransferase (COMT).^28,117,118 The abnormality in these enzymatic systems has been observed in AD patients, suggesting that targeting neurotransmitter-related enzymes would be a valuable therapeutic strategy.^28,117,118

AChE is a well-known and extensively studied factor in the pathology of AD (Fig. 4a). According to the “cholinergic hypothesis,” AD is characterized by a significant loss of cholinergic neurons and a decline in ACh levels in the brain, leading to memory loss and cognitive deficits.^28,122 As a result, AChE becomes a major target for developing therapeutic inhibitors. The structures of AChE and commonly prescribed AChE inhibitors, such as rivastigmine, galantamine, and donepezil, are presented in Fig. 4a.^28–32,123 Recombinant human AChE (rhAChE) contains several essential binding domains. The catalytically active site (CAS) is located at the bottom of a 20 Å deep, narrow binding gorge, and its anionic domain includes amino acid residues W86, Y130, Y337, and F338. The peripheral anionic site (PAS), positioned at the entrance of the binding gorge, comprises amino acid residues Y72, D74, Y124, W286, and Y341.^111,112 Current AChE inhibitors work by competitively impeding the enzyme's activity, either by blocking the CAS to prevent substrate interaction or by hindering the access to the active site through the PAS.^28–32 For example, donepezil interacts with both the CAS and the PAS of AChE, forming hydrogen bonds with water molecules that connect to the side chains of Y337 and Y341, and engaging in π–π interactions with the side chains of W86 and W286, as illustrated in Fig. 4a.¹¹² These interactions interfere the breakdown of ACh into acetate and choline, thereby increasing the availability of ACh and enhancing cholinergic neurotransmission.²⁸

Fig. 4 AChE, MAOB, and examples of their inhibitors. (a) Structure of AChE (PDB 4EY4 ¹¹¹), binding mode of donepezil at its active site (PDB 7E3H¹¹²), and three U.S. FDA-approved AChE inhibitors (donepezil, rivastigmine, and galantamine). The amino acid residues involved in donepezil binding are highlighted in red and depicted as sticks. (b) Structure of MAOB (PDB 1GOS¹¹³), interaction with selegiline at its catalytic domain (PDB 2BYB³³), and three representative inhibitors (lazabemide, selegiline, and safinamide). The amino acid residues involved in selegiline binding are indicated in red with sticks.

Additionally, the distinctive selectivity of AChE inhibitors against butyrylcholinesterase (BuChE) plays a significant role in their efficacy and suitability for different stages of AD.^124–126 Unlike AChE, BuChE shows weaker substrate preference due to the absence of several aromatic residues, retaining only one aspartate and tyrosine residue in its PAS. Interestingly, BuChE activity increases during the late stage of AD, particularly in neurodegenerative regions, which compensates for the decline in AChE activity.^124–126 Consequently, non-selective inhibitors may offer advantages in later stages of AD by targeting both enzymes, though they also carry a higher risk of gastrointestinal and cardiovascular side effects.^124–126 Currently developed AChE inhibitors exhibit varying selectivity for AChE and BuChE. For example, rivastigmine addresses both AChE and BuChE, while galantamine and donepezil demonstrate moderate and strong selectivity for AChE, respectively.^124–126

Along with the cholinergic hypothesis, the loss of dopaminergic and serotonergic neurotransmission represents another pathological factor in AD.^127–129 The degeneration of dopaminergic and serotonergic neurons leads to significant local inflammation and exacerbates age-related cell death, as observed in the AD-transgenic Tg2576 mice model.¹²⁷ This neuronal loss is associated with pathological symptoms, including impairments in hippocampal neuronal excitability, synaptic plasticity, memory, and reward performance.^127–129 Furthermore, the activation of enzymes that degrade dopamine and serotonin, such as MAO and COMT, has also been implicated in the pathology of AD.^{103,117,118,130} Consequently, numerous efforts have focused on targeting these enzymes. Recent studies have highlighted MAO inhibitors, such as lazabemide, selegiline, and safinamide, which are either U.S. FDA-approved or in clinical trials, as displayed in Fig. 4b.¹³¹ These inhibitors block the active site of MAO (primarily MAOB), which consists of two cavities: the substrate cavity near the flavin adenine dinucleotide (FAD) and the entrance cavity.¹¹³ For instance, selegiline interacts with the substrate/FAD binding site, including amino acid residues L171, C172, I199, Q206, Y326, F343, and Y398, through van der Waals, CH–π, and π–π interactions that are essential for binding.³³Selegiline then forms covalent bonds with FAD, irreversibly prohibiting substrate coordination and subsequent reactions, thereby alleviating the loss of dopamine and serotonin under pathological conditions (Fig. 4b).³³ Despite their potential to restore neurotransmission, these drugs cannot halt disease progression and provide only modest, short-term cognitive benefits, often accompanied by significant side effects.^28,132 While AChE and MAO inhibitors have been the cornerstone of AD treatment, these drawbacks underscore the urgent need for novel therapies. The inadequate efficacy and adverse effects of current treatments emphasize the necessity for disease-modifying therapies that can concurrently address multiple pathophysiological mechanisms of AD.

2.4 Other pathological features

In addition to the abovementioned pathological factors, neuroinflammation, mitochondrial dysfunction, and neuronal cell death are closely associated with the pathogenesis of AD (Fig. 1a).^4–7 These features are intricately connected to other pathological elements, such as Aβ and ROS, underscoring the multifaceted nature of the disease.^6–15 Under normal conditions, astrocytes and microglia play distinct yet complementary roles in maintaining homeostasis and managing neuroinflammation in the central nervous system (CNS). Astrocytes preserve the integrity of the blood–brain barrier (BBB), regulate cerebral blood flow, and release anti-inflammatory cytokines, contributing to a stable neural environment.^133–137 Meanwhile, microglia, the primary immune cells of the CNS, constantly monitor their microenvironment and secrete pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in response to injury or infection.^138,139 Together, these cells balance neuroprotection and tissue repair to support neural homeostasis. In AD, however, this balance is disrupted, leading to chronic neuroinflammation and neuronal death.

Aβ aggregates activate microglial receptors, such as Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), which stimulate the NLR family pyrin domain-containing 3 (NLRP3) inflammasome.^140–144 Once induced, the NLRP3 inflammasome promotes Aβ aggregation by serving as a nucleation platform for fibrillization. It also cleaves pro-caspase-1 into active caspase-1, which processes pro-interleukin-1β (pro-IL-1β) and pro-interleukin-18 (pro-IL-18) into their secretory forms.^140–144 The release of cytokines, such as IL-1β and TNF-α, amplifies inflammation and prompts astrocytes to secrete neurotoxic molecules, including ROS and complement components (e.g., complement component 3).^145,146 Simultaneously, sustained excitation of nuclear factor-κB (NF-κB) in astrocytes and microglia, triggered by Aβ and cytokine feedback loops, further elevates the production of pro-inflammatory mediators, including interleukin-6 (IL-6) and TNF-α, perpetuating inflammation and oxidative stress.¹⁴⁷ Chronic glial activation also impairs microcirculation and disrupts the BBB, exacerbating Aβ accumulation.^148,149 Through these interconnected mechanisms, astrocytes and microglia transition from protective roles to active contributors to neuronal damage, driving the progression of AD.

Therapeutic strategies to address astrocyte- or microglia-mediated neuroinflammation in AD primarily focus on modulating the NF-κB pathway, a central regulator of inflammatory responses. These strategies include developing antagonists for membrane receptors that activate NF-κB^150–153 and inhibitors of key intracellular signaling molecules within this pathway.^154–157 Complementary approaches aim to mitigate Aβ aggregation driven by neuroinflammation.^158–164 One promising method involves targeting the active NLRP3 inflammasome, which serves as a nucleation site for Aβ aggregation, thereby attenuating harmful effects of pro-inflammatory responses.^158–164 Another strategy focuses on enhancing microglial phagocytosis and autophagy to promote the clearance of Aβ aggregates, offering the dual benefit of reducing inflammation and preventing further neuronal damage.^165–169

Mitochondrial dysfunction is another critical pathological feature of AD, closely linked to disease progression through several key mechanisms. One major manifestation is the overproduction of ROS, which induces oxidative stress and damages cellular components, such as lipids, proteins, and DNA.¹⁷⁰ Bioenergetic deficits, such as reduced ATP production by disruptions in the electron transport chain (ETC), further compromise neuronal energy supply, ultimately leading to synaptic failure and cell death.^171,172 Mitochondrial DNA (mtDNA) degradation exacerbates these effects by impairing metabolic functions and repair mechanisms, intensifying cellular dysfunction.^173,174 A key contributor to mitochondrial dysfunction is the diminished clearance of damaged mitochondria, primarily regulated by the phosphatase and tensin homolog-induced kinase 1 (PINK1)-Parkin pathway.^175,176 This pathway is altered under pathogenic conditions due to the accumulation of hyperphosphorylated tau and Aβ, resulting in mitochondrial defection and increased cellular stress.^177,178 Additionally, the balance between mitochondrial fusion and fission is disturbed. Aβ and ROS overactivate dynamin-related protein 1 (Drp1), causing excessive mitochondrial fragmentation.^179–182 This unregulated cleavage compromises energy distribution, weakens synaptic function, and exacerbates neuronal damage.^179–182 Mitochondrial biogenesis is also hindered by oxidative stress and neuroinflammation, which suppress peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), thereby reducing the generation of functional mitochondria and worsening bioenergetic deficits.^183–185 Abnormal mitochondrial distribution, coupled with endoplasmic reticulum damage, further amplifies mitochondrial dysfunction, positioning it as a central driver of AD progression.^186,187

Strategies to address mitochondrial dysfunction in AD focus on reducing excess ROS, restoring mitochondrial function, and improving bioenergetics. To mitigate ROS overproduction, various therapeutic agents with antioxidant functional groups have been explored.^188–190 Efforts are also underway to restore mitochondrial biogenesis, dynamics, and mitophagy by targeting essential proteins involved in these processes, such as nicotinamide adenine dinucleotide-dependent deacetylase sirtuin-1 (SIRT1) and mammalian target of rapamycin (mTOR), which inhibits PGC-1α and the PINK1-Parkin signaling, respectively.^191–195 Furthermore, modulating mitochondrial bioenergetics by enhancing components critical to energy production in mitochondria, such as the ETC and Krebs cycle enzymes, may improve energy production in AD.^{187,196–201}

The interconnected pathological systems and signaling cascades drive apoptosis-mediated neuronal cell death through both intrinsic and extrinsic pathways.^202–204 Oxidative stress, mitochondrial dysfunction, and Aβ accumulation disrupt intracellular membranes, leading to the release of proteins, such as cytochrome c, into the cytosol.^202–206 This triggers the formation of the apoptosome complex and activates caspase-9, further exacerbating cell death and promoting mitochondrial permeabilization.²⁰⁵ Dysregulated survival pathways, including phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mTOR signaling network, further contribute to apoptosis by impairing protective mechanisms and initiating stress-induced responses.^15,207,208 Extracellular Aβ aggregates also activate cell surface death receptors (e.g., Fas or TNF), forming a death-inducing signaling complex that induces the extrinsic apoptotic pathway via caspase-8.^209,210 Caspase-8 either directly stimulates caspase-3 or amplifies the intrinsic pathway by cleaving the Bcl-2 homology 3 interaction domain death agonist (Bid) into truncated Bid (tBid), which further promotes apoptosis.^211,212 Neuroinflammation intensifies these effects via pathways, such as Janus kinase/signal transducer and activator of transcription (JAK-STAT), upregulating the expression of pro-inflammatory and pro-apoptotic genes. Concurrently, alterations in Ras/extracellular signal-regulated kinase (Ras/ERK) signaling compromise neuronal survival, which accelerates apoptosis-directed neurodegeneration.^213,214

Non-apoptotic cell death pathways also play a significant role in neurodegeneration associated with AD. Dysregulated autophagy, initiated by Aβ aggregates and impaired lysosomal function, leads to the accumulation of dysfunctional organelles and proteins, worsening neuronal damage.^215,216 Pyroptosis, an inflammatory form of cell death, is driven by the activation of the NLRP3 inflammasome, releasing pro-inflammatory cytokines and causing membrane damage via gasdermin-D-mediated pathway.^144,217 Cuprotosis, arising from imbalances in copper metabolism, disrupts the mitochondrial function, elevates the oxidative stress, and results in the misprocessing of APP, ultimately contributing to neuronal death and exacerbating Aβ pathology.²¹⁸ Ferroptosis, an iron-dependent cell death mechanism, is fueled by perturbed iron metabolism, oxidative stress, and lipid peroxidation, thereby promoting neuronal loss.^219,220 Collectively, these apoptotic and non-apoptotic pathways form a feedback loop of inflammation, mitochondrial dysfunction, and neuronal damage, which fosters the progression of AD. To regulate apoptosis, inhibitors confronting caspases-3, -8, and -9 are being actively investigated, with caspase-1 inhibitors to mitigate pyroptosis.^221–232 Additionally, metal chelators are being explored as a potential therapy to control metal ion dyshomeostasis-mediated cell death, such as cuprotosis and ferroptosis.^2,218,232 Taken together, neuroinflammation, mitochondrial damage, and neuronal cell death are increasingly recognized as key pathological targets in AD. In addition to the aggregation of Aβ, the dyshomeostasis of metal ions, and the dysfunction of enzymatic systems, these pathological features highlight the multifaceted nature of AD.^4–7 Ongoing research efforts are focused on developing therapeutic interventions to address these interconnected processes, as emphasized in recent literature.^6–15

3. Strategies to overcome the complex pathology of AD

Numerous single-target-directed drugs have been developed to address specific pathological factors associated with AD. These treatments have been evaluated at various clinical trial stages, highlighting their potential therapeutic roles within the AD landscape.^17,32,55,56 Despite extensive efforts, no single treatment has been able to effectively alter the progression of the disease, achieving limited success.^{3,18,20,21,25,41,59,77–79,233} This outcome underscores the complexity of AD, where multiple factors contribute to disease onset.¹⁶ Therefore, there is a critical need for therapeutics capable of targeting several pathological factors simultaneously.

Rational design strategies for multi-target-directed molecules involve combining two or more molecular frameworks, each with established properties, into a single molecular entity, as illustrated in Fig. 1b.^{35,126,234,235} This integration can be accomplished through three approaches: (i) linkage approach, where the original structures are connected via a linker; (ii) fusion approach, which directly joins two scaffolds without a linker; (iii) incorporation approach, where functional components are merged while preserving structural features essential for their activity.^{35,126,234,235} Achieving this multifunctionality requires a careful balance of scaffold properties while maintaining their functional integrity.^{35,126,234,235} These resulting compounds can then undergo structure–activity relationship (SAR) optimization to refine their physicochemical properties, target specificity, and BBB permeability to maximize their therapeutic potential.^{35,126,234,235}

Multifunctional molecules developed to address Aβ aggregation often use frameworks inspired by known imaging agents, e.g., thioflavin-T (ThT), polyphenols, and peptide-based inhibitors.^{35,126,234,235} These scaffolds are strategically modified to strengthen interactions with the regions of Aβ, including the self-recognition and C-terminal regions, both of which play a crucial role in Aβ aggregation.³⁹ By targeting these regions, the molecules can disrupt aggregation pathways and prevent the formation of toxic oligomers and fibrils.³⁹ These compounds typically incorporate hydrophobic features to enhance binding to Aβ′s hydrophobic domains, as well as π–π stacking or hydrogen bonding capabilities to interact with multiple amino acid residues within the peptide.³⁹

MTDLs designed for confronting Aβ aggregation and metal ion dyshomeostasis simultaneously require the incorporation of nitrogen, oxygen, or sulfur donor atoms for metal chelation into Aβ-binding frameworks to achieve dual function.^35,94–96 By precisely tuning dissociation constant (K_d) values and metal-binding geometries, these chelators can prohibit interactions between metal ions and Aβ without disrupting systemic metal ion homeostasis or interfering with the function of metalloenzymes. Additionally, MTDLs can be optimized to block vacant coordination sites within metal-binding domains, thereby preventing redox reactions that produce ROS. Moreover, these molecules can modulate the redox potential of the metal center to mitigate oxidative stress.

To regulate enzyme activity, rational strategies focus on targeting the active sites of enzymes.^{54,104,108,111,112} A common approach is competitive inhibition, where molecules are designed to mimic natural substrates, enabling them to occupy the active sites of enzymes and block their substrate binding. By integrating computational modeling and crystallographic studies, the development of highly selective and effective MTDLs capable of adjusting enzyme activity is facilitated. Rational design often utilizes backbones from natural products, polyphenols, or established inhibitors, employing interactions, such as hydrogen bonding, π–π stacking, and hydrophobic contacts, to acquire high specificity.^236,237

Multifunctionality can be achieved by linking or incorporating key moieties addressing multiple pathways into a single molecular framework. While research on Aβ aggregation remains a central focus, extensive efforts are also being directed toward combining two or more distinct structural motifs within a single molecule to simultaneously target alternative pathological pathways.^{35,126,234,235} These efforts include inhibiting enzymes, such as BACE1, MMP9, LOXL2, AChE, and MAOB, as well as modulating their interactions with metal ions.^{35,126,234,235} Such approaches, which have been comprehensively reviewed in several studies, highlight the versatility and therapeutic promise of MTDLs.^{35,126,234,235} The following sections underscore the critical role of SAR studies, with an emphasis on the development of multi-target-directed compounds that focus on Aβ aggregation with other interconnected pathological factors of AD.

3.1 Chemical reagents capable of controlling Aβ and BACE1

Aβ remains a critical biomarker in AD therapy due to its association with the disease's pathological features and its involvement in neuroinflammation and cognitive decline.¹ Consequently, regulating BACE1 activity, the primary enzyme responsible for Aβ production, has become a major focus in the development of AD therapeutics.^50,238 Single-target-directed therapies, however, have proven insufficient in slowing disease progression. This has led to a shift in research towards strategies that regulate both the accumulation and production of Aβ to achieve a synergistic clinical effect.^233,238 Therapeutic interventions that integrate multiple characteristics can effectively form hydrophobic interactions or hydrogen bonds within the catalytic cleft and aspartate dyad to target BACE1 efficiently. Additionally, these structures can be designed to establish hydrophilic and hydrophobic contacts with Aβ, thereby inhibiting its aggregation. Current approaches with the above factors are widely being explored to address both Aβ and BACE1 using peptides,²³⁹ natural products,^240,241 and modifications of existing therapeutic frameworks.^242,243

Peptides naturally contain polar groups that can be modified to improve their ability to cross the BBB and interact efficiently with target enzymes. To optimize these properties, Singh and coworkers designed peptides with hydrophobic groups added at both the N- and C-termini.²³⁹ Proline was used as the template to regulate surface area and structural flexibility, with a glycine–proline–alanine (GPA) tripeptide as the model backbone.²³⁹ Various derivatives, each featuring different functional groups at the N- and C-termini, were synthesized to achieve an optimal balance between hydrophobicity and hydrophilicity. Among these derivatives, GPA-1 and GPA-2 (shown in Fig. 5a) exhibited significant in vivo efficacy, effectively inhibiting both the activity of BACE1 and the aggregation of Aβ₄₂.²³⁹ Both peptides displayed potent ID₅₀ values of 20 nM against BACE1.²³⁹ Additionally, GPA-1 and GPA-2 suppressed the formation of β-sheet-rich Aβ₄₂ aggregates by 54% and 34%, respectively.²³⁹ Pretreatment with these peptides also alleviated memory deficits in a mouse model with scopolamine-induced memory impairment, as presented in Fig. 5a.²³⁹ Behavioral assessments, including the Morris water maze, revealed a significant reduction in latency time to reach the platform, while the passive avoidance task demonstrated decreased latency in reaching the shock-free area.²³⁹ Furthermore, peptide-based strategies have been applied to block the β-cleavage site of APP in in vivo systems.²⁴⁴

Fig. 5 Examples of chemical reagents controlling Aβ and BACE1. (a) Small peptide-based compounds (GPA-1 and GPA-2) and their effects on the cognition of mice models after inducing impaired memory with scopolamine on days 12 to 14. GPA-1, NH₂-Gly-Pro-Ala-OMe; GPA-2, Cbz-Gly-Pro-Ala-O-cinnamyl. Reproduced with permission from ref. 239. Copyright© 2024 American Chemical Society. (b) A flavonoid-based natural product (YCC31) and its inhibitory impact on the activity of BACE1 and the aggregation of Aβ₄₂. The interactions between YCC31 and BACE1 at the active site, the morphology of the resultant Aβ₄₂ species, and its inhibitory effect on Aβ₄₂ aggregation were analyzed by MD simulations, TEM, and the ThT assay, respectively. YCC31, quercetin-3-O-[β-D-glucopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→6)-O-β-D-glucopyranoside]. Reproduced with permission from ref. 240. Copyright© 2024 Elsevier. (c) A phenothiazine-derived compound (19c) and its effect against the activity of BACE1 and the aggregation of Aβ₄₀. The interactions between 19c and BACE1 at the active site were obtained from docking studies and its inhibitory effect on the formation of β-sheet-rich Aβ₄₀ aggregates was quantitatively measured by the ThT assay. 19c, 4-amino-N-[(4-chlorobenzyl)]-2-[methyl({2-[2-(10H-phenothiazin-10-yl)ethoxy]ethyl})amino]butanamide. Reproduced with permission from ref. 243. Copyright© 2023 Elsevier.

The use of natural products as therapeutic candidates has garnered attention for decades due to their proven efficacy, safety, and increased molecular rigidity compared to synthetic compound libraries, particularly for addressing protein–protein interactions.²⁴⁵ Among these natural compounds, flavonoids are notable for their ability to interact with multiple biological targets through hydrogen bonding and π–π stacking interactions.^240,246,247 Building on these properties, Yang and coworkers discovered a natural flavonoid–carbohydrate conjugate, YCC31, which exhibited significant potential for dual inhibition (Fig. 5b). YCC31 reduced Aβ production in 7PA2 cells by modulating the activity of BACE1.²⁴⁰ The compound forms hydrogen bonds with key amino acid residues G95, F169, R189, Y259, K285, and D289 within BACE1 through its hydroxyl groups and ether group oxygen atom. Additionally, its phenyl group participates in CH–π interactions with T292, effectively preventing substrate access and reducing Aβ production, as depicted in Fig. 5b.²⁴⁰ In addition to BACE1 inhibition, YCC31 demonstrated dose-dependent regulation of Aβ₄₂ aggregation, as analyzed by transmission electron microscopy (TEM), ThT assay, and molecular dynamics (MD) simulations.²⁴⁰ The supramolecular equivalent of YCC31 suppressed the fibrillization of Aβ₄₂, generating short fibrils by interacting with amino acid residues D22, S26, N27, and K28, which are crucial for the salt bridging that stabilizes fibrillar structures.²⁴⁰ Moreover, YCC31 decreased the production of ROS and nitric oxide (NO) induced by Aβ-mediated activation of microglia, thereby mitigating oxidative stress and neuroinflammation.²⁴⁰

Modifying existing scaffolds has emerged as a promising approach to accelerate the drug discovery process.²⁴⁸ One notable example is N-benzylbutanamide.²⁴³ Bajda and coworkers developed a phenothiazine derivative, 19c, which exhibited the highest hBACE1 inhibitory activity (IC₅₀ = 1.6 μM) and anti-aggregation effects against Aβ₄₀ (99% inhibition at 10 μM), as described in Fig. 5c.²⁴³ The inhibition of hBACE1 by 19c is attributed to its ability to block both substrate entry and catalytic proteolysis. The compound interacts with the S2 and S2′ pockets via CH–π interactions with Y71 and a π–oxygen bond with Y198, while forming a cation–π interaction with R128 in the S3′ pocket. Additionally, its amide group establishes a hydrogen bond with the main chain of T72, and its γ-amino group engages in a network of bonds, creating two hydrogen bonds with the side chains of T72 and T329 and an ionic interaction with D328.²⁴³ The phenothiazine moiety of 19c fits into the S3 and S4 pockets of BACE1, accompanying in hydrophobic contacts with Q73 and I110.²⁴³ Moreover, 19c prevented Aβ₄₀ aggregation by 86%, significantly decreasing both elongation and nucleation rates, as measured by the ThT assay (Fig. 5c). These results indicate that 19c directly associates with fibrillar species to suppress elongation and with soluble nucleic, oligomeric, and prefibrillar forms of Aβ₄₀ during the early stages of polymerization. Beyond its in vitro effects, 19c demonstrated statistically significant anti-amnesic properties and improved non-spatial (contextual and recognition) memory in a scopolamine-induced amnesia mouse model.²⁴³ Overall, these findings underscore the potential of multi-targeting strategies in developing effective AD therapeutics, particularly those involving precise molecular interactions with BACE1 and Aβ.

3.2 Chemical reagents capable of regulating Aβ, metal ions, and ROS

Metal ions, such as Cu(I/II), Zn(II), and Fe(II/III) can coordinate to Aβ to form metal–Aβ complexes.^82,249 The non-specific removal of these metal ions, however, has been unsuccessful and presents several challenges.^25,77–79 As a result, strategies promising alternatives to non-selective metal ion chelation. To achieve this, numerous aspects of each component must be carefully considered. Chemical reagents should incorporate either a redox-active backbone, a metal-chelation moiety, or both to effectively mitigate oxidative stress.^25,77 In addition, the structure should include functional groups capable of forming hydrogen bonds and hydrophobic interactions to regulate Aβ aggregation.^35,234,235 For these aspects, peptides have been developed.^250–256 Sun and coworkers discovered that integrating the Aβ self-recognition site (LVFFA) and a metal-chelating tripeptide (RTH) efficiently modulates metal-mediated Aβ aggregation and ROS generation (Fig. 6a).^250–252 Building on this concept, a D-enantiomeric peptide, RTHLVFFARK-NH₂ (rk10), was rationally designed and compared to its previously reported L-enantiomer, RK10. The rk10 peptide demonstrated the ability to form stable metal coordination with the amino terminal Cu(II)- and Ni(II)-binding (ATCUN) motif, exhibiting a K_d value of 28 nM, comparable to that of Aβ (log K_Cu(II) = 10). This Cu(II) chelation prevented redox reactions and excessive ROS production by Cu(II)–Aβ complexes.²⁵⁰ As shown in Fig. 6a, rk10 treatment decreased ROS generation to half the level observed in the Cu(II)–Aβ control without peptide treatment.²⁵⁰ Furthermore, rk10 disrupted the twisted and elongated fibrillar network, suppressing fibril growth and redirecting the aggregation pathway to yield amorphous aggregates, as visualized by atomic force microscopy (AFM).²⁵⁰ A similar trend was observed in the presence of Cu(II), where rk10 addition noticeably decreased the formation of visible structures, compared to RK10.²⁵⁰ These findings suggest that while rk10 possesses metal–chelation properties similar to RK10 (K_d = 31 nM), its enhanced interactions with Aβ further improve the inhibitory effects on Aβ fibrillization and Cu(II)–Aβ-induced ROS generation.^250,252

Fig. 6 Examples of chemical reagents regulating Aβ peptides and metal ions. (a) Structure of the D-enantiomeric decapeptide RTHLVFFARK-NH₂ (rk10) and inhibitory effects of rk10 and its L-enantiomer (RK10) on the generation of ROS and metal-free and Cu(II)–Aβ₄₂ aggregation. The ROS level produced by Cu(II)–Aβ₄₂ in the absence and presence of rk10 or RK10 in SH-SY5Y cell was measured by a fluorescence assay with 2′,7′-dichlorofluorescin diacetate (DCFH-DA). The statistical significance level is expressed by the pound (control as Aβ₄₂ + Cu(II), ^###P < 0.001) and asterisk (control as Aβ₄₂ + Cu(II) + RK10, *P < 0.05). The morphology of Aβ₄₂ aggregates generated with and without Cu(II) and rk10 or RK10 was analyzed by AFM. Reproduced with permission from ref. 250. Copyright© 2019 American Chemical Society. (b) Structure of PPD and its impact on the aggregation of both metal-free and metal-added Aβ₄₀. The size distribution of Aβ₄₀ aggregates produced with and without PPD or metal ions was investigated by gel/western blot. The formation of the covalent adduct between Aβ₄₀ and benzoquinone was detected by ESI–MS. PPD, p-phenylenediamine. Reproduced with permission from ref. 257. Copyright© 2020 American Chemical Society. (c) Structure of L1 and its influence on Cu(II)-added Aβ₄₀ aggregation. The size distribution and morphology of the resultant Aβ₄₀ aggregates produced with and without L1 or Cu(II) were investigated by gel/western blot and TEM, respectively. Chemical modifications onto the Cu(II)-coordination sphere of Aβ₄₀ by L1 were monitored by ESI–MS. L1, N¹,N¹-dimethyl-N⁴-(thiophen-2-ylmethyl)benzene-1,4-diamine. Reproduced with permission from ref. 258. Copyright© 2020 United States National Academy of Sciences.

An alternative strategy in AD research involves using small molecules that chemically modify Aβ, altering its aggregation pathways. A series of redox-active aromatic compounds has demonstrated reactivities towards metal-free Aβ, metal-bound Aβ, and free organic radicals.²⁵⁷ Among these, p-phenylenediamine (PPD; Fig. 6b) and its derivatives have emerged as promising therapeutic candidates due to their structural simplicity, redox capability, and amphiphilicity.^259,260PPD, with two electron-donating groups at the para position, lowered the redox potential and enhanced its ROS scavenging capacity.^261,262 Furthermore, PPD was shown to modulate Aβ aggregation, resulting in smaller fibrils and amorphous aggregates, which appeared as smearing bands in the 4–270 kDa range in gel electrophoresis with western blotting (gel/western blot) analyses (Fig. 6b).²⁵⁷ A similar trend was observed in the presence of Cu(II) or Zn(II), indicating that PPD further perturbed metal–Aβ aggregation pathways.²⁵⁷ Mechanistic details revealed that PPD undergoes oxidation to yield benzoquinone-diimine (BQDI) or benzoquinone (BQ)²⁵⁷ during which the M35 residue of Aβ is oxidized.²⁵⁷ In addition, BQ formed covalent adducts with Aβ through primary amino groups, such as K16 (Fig. 6b).^257,263,264 This complexation captured Aβ in a relatively compact conformation, likely preventing its involvement in Aβ fibrillization.²⁶⁵ Supported by these in vitro results, PPD significantly reduced cerebral and hippocampal Aβ deposits and improved cognitive function in 5xFAD transgenic mice.²⁵⁷

The structural similarity of N,N-dimethylaniline (DMA) to PPD imparts comparable properties, enabling DMA to interact with both metal-free Aβ and metal–Aβ, and participate in one- or two-electron oxidation processes, thereby contributing to ROS scavenging.^266,267 Building on this concept, a derivative named L1 (presented in Fig. 6c), featuring a bidentate ligand attached to the DMA backbone, was developed to directly modify the Cu(II)-coordination sphere in Aβ through copper–O₂ chemistry.²⁵⁸ Notably, the H14 residue was modulated when Cu(II)–Aβ was incubated with L1 under aerobic conditions, resulting in mass increments of 16 Da and 96 Da in Aβ, corresponding to oxidation and covalent adduct formation, respectively.²⁵⁸ Since H14 is associated with coordinating both Cu(I) and Cu(II) in Aβ, this chemical transformation effectively disrupted the aggregation of metal–Aβ complexes.²⁵⁸ Moreover, L1 inhibited the aggregation of Cu(II)–Aβ₄₀ by altering the morphology of Aβ aggregates into shorter fibrils, suppressed H₂O₂ production from the redox cycling of Cu(I/II)–Aβ, and reduced cytotoxicity (Fig. 6c).²⁵⁸

Overall, several small molecules have been developed to address metal-induced Aβ aggregation through three primary mechanisms: (i) directly interacting with both metal ions and Aβ, (ii) chemically modifying Aβ, and (iii) disrupting the coordination sphere of metal–Aβ complexes. These strategies can precisely redirect the binding pattern of redox-active metal ions to Aβ, thereby preventing ROS production. They further suggest that shifting beyond traditional approaches, which solely focus on chelating metal ions, to simultaneously targeting both metal–Aβ interactions and ROS may present a more effective strategy for developing novel AD treatments.

3.3 Chemical reagents capable of modulating Aβ and metalloenzymes

Metal ion dyshomeostasis and the resultant dysregulation of metalloenzymes are emerging as promising therapeutic targets for AD.^{98,104–107,120,268–272} Among these, MMP9 and LOXL2 have been implicated in the pathogenesis of neurodegenerative diseases due to their involvement in Aβ aggregation.^104–107 Consequently, there is growing interest in developing therapies that simultaneously address MMP9 or LOXL2 while inhibiting Aβ aggregation as a combined approach to enhance therapeutic efficacy.

As part of metalloenzymes, regulating the activity of MMP9 and LOXL2 requires the inclusion of a metal-coordination motif, with functional groups capable of disrupting Aβ self-assembly. Building on this concept, several polyphenol-based natural products have been investigated for their regulatory properties against MMP9.^273,274 Among them, doxycycline (DOX; Fig. 7a), a tetracycline derivative, is a well-known antibiotic that also inhibits MMP9 and reduces neuronal damage in cases of focal brain ischemia.²⁷⁸ This prospective dual-modulatory effect has advanced DOX to phase II clinical trials.²⁷⁹ Kaur and coworkers elucidated the binding mode and antagonistic mechanism of DOX against MMP9 through docking analysis.²⁸⁰DOX primarily interacts with the catalytic domain of MMP9 by forming hydrogen bonds with amino acid residues E402, E416, and L418, and through hydrophobic interactions, such as CH–π and π–π interactions with amino acid residues H401, P421, M422, Y423, and R424 (highlighted green in Fig. 7b).²⁸⁰ Additionally, the coordination of DOX to Zn(II) through the hydroxyl group on its D ring further stabilizes its complexation at the active site, thereby preventing ligand association and catalytic hydrolysis.²⁸⁰

Fig. 7 Examples of chemical reagents controlling Aβ and MMP9 or LOXL2. (a) Structure of doxycycline [DOX; (4S,4aR,5S,5aR,6R,12aS)-4-(dimethylamino)-3,5,10,12,12a-pentahydroxy-6-methyl-1,11-dioxo-1,4,4a,5,5a,6,11,12a-octahydrotetracene-2-carboxamide]. (b) Intermolecular interactions between MMP9 (PDB 1L6J⁷⁰) and DOX. The Zn(II)-coordination and DOX-interacting residues are indicated as sticks, and highlighted in red and green, respectively. (c) Interactions of DOX with Aβ₄₂ fibrils (PDB 2MXU). The morphology of the resultant Aβ₄₂ species was analyzed with TEM. Reproduced with permission from ref. 275 and 276. Copyright© 2019 Multidisciplinary Digital Publishing Institute and 2001 John Wiley and Sons. (d) Structure of curcumin (CUR). CUR, (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione. (e) Intermolecular interactions between LOXL2 (PDB 5ZE3 ⁷¹) and CUR. The Cu(II)-coordination and CUR-interacting residues are shown as sticks, and depicted in red and green, respectively. (f) Interactions of CUR with Aβ₄₂ fibrils (PDB 2MXU). The formation of the smaller Aβ₄₂ species was detected by TEM. Reproduced with permission from ref. 275 and 277. Copyright© 2019 Multidisciplinary Digital Publishing Institute and 2017 Elsevier.

Recent studies have also elucidated that DOX directly associates with amyloidogenic fibrils, including those produced by Aβ, and disassembles pre-formed aggregates.^{275,276,279,281,282} As depicted in Fig. 7c, DOX engages in hydrophobic interactions with key amino acid residues of Aβ₄₂ fibrils, such as L17, F19, I32, and L34, which are critical for self-recognition and fibril stabilization (Fig. 2b).²⁷⁵ Morphological analysis revealed that DOX treatment significantly reduced fibril generation and promoted degradation.²⁷⁶ In addition to its anti-aggregation properties, DOX has been shown to scavenge ROS and other oxidants, such as superoxide anion radicals (O₂˙⁻) and lipid peroxides, diminishing the production of cytotoxic malondialdehyde-acetaldehyde–protein adducts mediated by redox reactions.²⁸³ These antioxidant properties of DOX contribute to its anti-inflammatory effects and its ability to alleviate oxidative stress in AD.²⁸³

Recent studies have shown that polyphenols can also regulate the activity of LOXL2, highlighting their potential as therapeutic agents.^275,277,284 Based on these findings, there is growing interest in identifying noble polyphenols that can simultaneously inhibit the activity of LOXL2 and the aggregation of Aβ. Curcumin (CUR; Fig. 7d), a natural polyphenol found in Curcuma longa plants, has gained recognition as a promising candidate for AD treatment due to its ability to target multiple pathological factors, including LOXL2 and Aβ.^275,277,284CUR modulated LOXL2 through mechanisms similar to those of DOX, binding directly to the catalytic domain and preventing ligand complexation and catalytic activity.²⁸⁴ This interaction involves hydrogen bonding between the amide backbone of P716 and a water molecule, which forms a hydrogen bonding network with the side chain of S512, along with hydrophobic interactions with the side chains of V713 and F718, as displayed in Fig. 7e.²⁸⁴ This binding mode resulted in a stable interaction, as confirmed by root mean square deviation (RMSD) analysis.²⁸⁴

Similar to DOX, CUR maintained continuous contact with Aβ fibrils during MD simulations, interacting with the central hydrophobic core of Aβ fibrils, including amino acid residues L17, F19, I32, and L34. CUR also disrupted the salt bridge formed by the side chain of D23. These interactions prohibited the aggregation of Aβ and destabilized fibril structures, leading to shorter, fragmented fibrils, as illustrated in Fig. 7f.²⁷⁵ In addition, CUR has been shown to inhibit the activity and expression of MMP9.²⁸⁵ Furthermore, CUR can chelate several metal ions, such as Cu(II), Fe(II), and Zn(II), through its α,β-unsaturated β-diketo moiety, thereby contributing to the reduction of ROS.^286,287 Collectively, addressing metalloenzymes, e.g., MMP9 and LOXL2, using multi-target-directed strategies offers a promising approach to enhancing specificity and therapeutic efficiency in AD treatment. Further research focusing on optimizing polyphenol backbones or modifications could significantly advance the development of AD therapeutics.

3.4 Chemical reagents capable of regulating metal-free Aβ, metal-bound Aβ, ROS, and AChE

The dysregulation of ACh, driven by the upregulation of AChE in senile plaques, is well-known to disrupt cholinergic neurotransmission and contributes to cognitive deficits in AD.^35,288 Despite the development of numerous AChE inhibitors, their efficacy has not met clinical expectations.^28–32 As a result, recent research has shifted towards multivalent therapeutic approaches that target multiple pathological factors to maximize efficacy while minimizing side effects.^{101,289–291}

For AChE inhibition, it is essential to prevent substrate binding to the active site by blocking the CAS, PAS, or both, thereby interrupting enzymatic activity mediated by the catalytic triad. Simultaneously, the molecule should incorporate features that enable interactions with Aβ, such as hydrogen bonding and hydrophobic properties. Flavonoid derivatives have shown promise in modulating several pathological factors, including ROS, metal-free and metal-bound Aβ, and AChE.^289,290,292 To enhance multi-targeting capabilities, structural modifications on the isoflavone or flavone core have been explored, as depicted in Fig. 8a.^289,290,292 These modifications increased the reactivity of flavonoid by incorporating multiple hydroxyl groups that disrupt substrate complexation at the AChE active site through hydrogen bonding. Additionally, the hydrophobic A and C rings of flavonoids interacted with aromatic side chains, such as W86, F295, and Y341, within the hydrophobic binding pocket (Fig. 8b).²⁹²

Fig. 8 Examples of chemical reagents regulating metal-free or metal-bound Aβ and AChE. (a) Structures of flavonoids. Isoflavone, 3-phenyl-4H-chromen-4-one; isoflavone-1, 3-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one; isoflavone-2, 5,6,7-trihydroxy-3-phenyl-4H-chromen-4-one; isoflavone-3, 3-(3,4-dihydroxyphenyl)-5,6,7-trihydroxy-4H-chromen-4-one; flavone, 2-phenyl-4H-chromen-4-one; flavone-1, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one; flavone-2, 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one; flavone-3, 2-(4-hydroxyphenyl)-3,5,7-trihydroxy-4H-chromen-4-one. (b) Intermolecular interactions between the flavonoids and AChE. Reproduced with permission from ref. 289 and 292. Copyright© 2023 Royal Society of Chemistry and 2020 Royal Society of Chemistry. (c) Impact of isoflavone-3 on the formation of metal-free Aβ₄₂ and metal–Aβ₄₂ aggregates. The morphology of the resultant Aβ₄₂ species was analyzed by TEM. Reproduced with permission from ref. 289. Copyright© 2023 Royal Society of Chemistry. (d) Structures of emodin and its derivatives with different functional groups at the R position. Emodin, 1,3,8-trihydroxy-6-methylanthracene-9,10-dione; emodin-1, 1,8-dihydroxy-3-methyl-6-(2-(piperidin-1-yl)ethoxy)anthracene-9,10-dione; emodin-2, 1,8-dihydroxy-3-methyl-6-(2-(pyrrolidin-1-yl)ethoxy)anthracene-9,10-dione; emodin-3, 1,8-dihydroxy-3-methyl-6-(2-(dimethylamino)ethoxy)anthracene-9,10-dione; emodin-4, 1,8-dihydroxy-3-methyl-6-(2-morpholinoethoxy)anthracene-9,10-dione. (e) Binding of emodin-1 against AChE. Reproduced with permission from ref. 291. Copyright© 2024 Elsevier. (f) Effect of emodin on Aβ₄₂ aggregation. The inhibitory activity of emodin on the production of β-sheet-rich Aβ₄₂ aggregates was quantitatively measured by the ThT assay. Reproduced with permission from ref. 101. Copyright© 2021 John Wiley and Sons. (g) Impact of emodin-1 and emodin-2 on Cu(II)-induced Aβ₄₂ aggregation. The influence of emodin-1 and emodin-2 on the generation of β-sheet-rich Cu(II)–Aβ₄₂ aggregates was analyzed by the ThT assay. Reproduced with permission from ref. 293. Copyright© 2023 Springer Nature.

Among these derivatives, a rationally designed and optimized flavonoid, isoflavone-3 (shown in Fig. 8a), which contains five hydroxyl groups on the isoflavone backbone, exhibited the highest reactivity.^289,290 As illustrated in Fig. 8b, the 5-OH group on the A ring formed hydrogen bonds with the backbone carbonyl group of G120–G121 and the hydroxyl group of S203. Additionally, the 3-OH group on the B ring generated a hydrogen bond with the backbone amide group between I294 and F295, while the O1 donor atom on the C ring constructed a hydrogen bond with the hydroxyl group of Y337.^289,294 These interactions effectively blocked the cavity located at the AChE active site, inhibiting the enzyme's function with an IC₅₀ value of 0.19 μM.²⁸⁹

Isoflavone-3 significantly modulated the oligomerization and fibrillization of both metal-free Aβ and metal–Aβ. As shown resulted in the formation of shorter and thinner fibrils, whereas in the presence of Cu(II) or Zn(II), it produced fragmented fibrils in Fig. 8c, the addition of isoflavone-3 to metal-free Aβ₄₂ with a mixture of amorphous and fibrillary aggregates.²⁸⁹ These alterations in aggregation were mediated by the interaction of isoflavone-3 with key regions of Aβ₄₂, including the β-turn motif (V12–Q15), self-recognition site (L17–A21), and the C-terminal region (I32–A42), which are critical for Aβ₄₂ fibrillization. Additionally, isoflavone-3 promoted the oxidation of H13, H14, and M35 in the presence of Cu(II), thereby disrupting metal coordination and the hydrophobic interactions between Aβ peptides, which influenced their aggregation behavior.²⁸⁹

The rational design of isoflavone-3 incorporated three hydroxyl groups at the C5–C7 positions on the A ring, a catechol moiety on the B ring, and translocation of the B ring from C2 to C3 on the C ring. This structural modification lowered its redox potential to 1.0 V (vs. SHE), enhancing its scavenging capability against free organic radicals.²⁸⁹ Overall, flavonoids like isoflavone-3 represent a promising platform for developing multifunctional therapeutics for AD by confronting multiple pathological features, including Aβ aggregation, metal ion dyshomeostasis, cholinergic dysfunction, and oxidative stress.

Several studies have suggested that anthraquinone compounds can alleviate AD symptoms by deactivating AChE through simultaneous targeting of the CAS and PAS, while also exhibiting antioxidant and anti-Aβ aggregation properties.^101,291Emodin (Fig. 8d), a natural anthraquinone-based polyphenol used in traditional Chinese medicine, is a well-researched chemical reagent that addresses multiple pathological factors in AD.^{101,291,295–297} Numerous structural modifications have been made to the emodin backbone to improve its dual activity against AChE and Aβ.²⁹¹ Among these derivatives, emodin-1 (illustrated in Fig. 8d) has demonstrated the most potent AChE inhibitory activity, with an IC₅₀ value of 67 nM.²⁹¹ The anthraquinone backbone of emodin-1 bound to PAS through CH–π interactions with W279, while additional hydrogen bonds, hydrophobic interactions, and π–π interactions occurred with F331.²⁹¹ Furthermore, functional groups, such as piperidine and pyrrolidine rings, formed CH–π interactions with W84 and H440, effectively blocking the CAS, as displayed in Fig. 8e.²⁹¹ These interactions at both the CAS and the PAS contributed to the efficient downregulation of AChE, reducing the degradation of ACh and thereby preserving cholinergic function.

Emodin has also been reported to modulate and inhibit Aβ₄₂ aggregation, both in the absence and presence of Cu(II). As presented in Fig. 8f, emodin reduced the β-sheet-rich aggregation of Aβ₄₂ by up to 80% in a dose-dependent manner,¹⁰¹ which is attributed to interactions between emodin's anthraquinone backbone and the V18 and F19 residues of Aβ, which form the self-recognition site.¹⁰¹Emodin derivatives with various functional groups have demonstrated similar modulatory effects on Aβ₄₂ aggregation, with emodin-1 achieving an inhibition ratio of ca. 76%.²⁹³ A comparable trend was observed in the presence of Cu(II), where significant reductions in β-sheet-rich aggregates were noted in samples treated with emodin-1 and emodin-2 (Fig. 8g).²⁹³ This effect is likely due to the interaction of emodin-1–4 (Fig. 8d) with the hydrophobic surface of the Aβ₄₂ helix in the N-terminal region, potentially stabilizing the α-helix and reducing the formation of oligomers and fibrils.²⁹³ Additionally, emodin possessed chelation properties that can regulate metal ion dyshomeostasis and oxidative stress. Treatment with emodin-1–4 dramatically decreased the formation of hydroxyl radicals (˙OH) and O₂˙⁻, compared to controls with Cu(II) alone.²⁹¹ These findings suggest that emodin derivatives display multiple reactivities towards pathological factors of AD, including metal chelation, oxidative stress modulation, Aβ aggregation control, and AChE inhibition, indicating their potential as promising MTDLs for AD treatment.^291,293

3.5 Chemical reagents capable of controlling Aβ and MAOB

The loss of dopaminergic and serotonergic neurotransmission in AD can be triggered by the increased activity of MAOB.^298,299 Inhibitors of MAOB, such as lazabemide, selegiline, and safinamide (Fig. 4b), have been shown to improve cognitive deficits and slow the progression of AD, making them therapeutically relevant candidates for anti-Alzheimer therapies.^300,301 Given the limitations in efficacy and potential side effects of MAOB inhibitors, there has been an increasing focus on targeting additional AD pathological features, such as Aβ aggregation and elevated oxidative stress, simultaneously. Several developmental strategies have been employed to achieve this dual functionality, enabling interactions with the active site of MAOB while possessing structural moieties to address Aβ aggregation. For example, modifications of existing therapeutics are emerging as viable candidates for this approach.

Rasagiline (Fig. 9a), a prominent MAOB inhibitor, is currently in phase II clinical trials for AD due to its dual ability to control Aβ aggregation through its 2,3-dihydro-1H-indene moieties.^304,305 Building on this framework, Xie and coworkers developed a selective and efficient MAOB inhibitor with the added capacity to target Aβ aggregation by combining the structural advantages of another MAO inhibitor, clorgyline, resulting in rasagiline–clorgyline conjugates.^302,306 Among the synthesized conjugates, as described in Fig. 9a, RC-6j, which features a five-carbon linker, exhibited the highest modulatory potency and selectivity, with an IC₅₀ of 4.0 nM and a selectivity index greater than 25 000.³⁰²RC-6j inhibited MAOB by competitive binding at the active site, interacting with the FAD cofactor and establishing a CH–π interaction with Y435 in the substrate cavity. Compared to rasagiline, the chromanone moiety of RC-6j occupied the entrance cavity, which is composed of amino acid residues W119, F168, L171, C172, I198, I199, I316, and Y326, through van der Waals and hydrophobic interactions, thereby enhancing the binding affinity.³⁰² In addition to MAOB inhibition, RC-6j also prevented Aβ₄₂ aggregation, significantly reducing β-sheet-rich structures by 40% and decreasing overall aggregation (Fig. 9a).^302,307 Furthermore, RC-6j mitigated neuronal damage caused by oxidative stress induced by 6-hydroxydopamine, a ROS generator that disrupts mitochondrial function through auto-oxidation.³⁰² These findings suggest that RC-6j is a promising MTDL for simultaneously controlling MAOB activity, Aβ aggregation, and oxidative stress in AD treatment.

Fig. 9 Examples of chemical reagents controlling Aβ and MAOB. (a) Structures of rasagiline, clorgyline, and RC-6j. The inhibitory effect of RC-6j against the activity of MAOB was represented in Lineweaver–Burk reciprocal plots, with its impact on the formation Aβ₄₂ aggregates. The catalytic rate of MAOB was measured through the amount of H₂O₂ generated by catalytic oxidation of p-tyramine, observed by the Amplex Red H₂O₂/peroxidase assay. The morphology of the resultant Aβ₄₂ species was monitored by TEM. RC-6j, 7-((5-(methyl(prop-2-yn-1-yl)amino)pentyl)oxy)chroman-4-one. Reproduced with permission from ref. 302. Copyright© 2020 Elsevier. (b) Structures of Schiff base derivatives and 3b, binding mode of 3b towards MAOB, and its effects on Cu(II)-added Aβ₄₂ aggregation. The morphology of the Cu(II)-induced Aβ₄₂ aggregates produced with and without 3b was investigated by TEM. 3b, 2,2′-((1E,1′E)-hydrazine-1,2-diylidenebis(methanylylidene))diphenol. Reproduced with permission from ref. 303. Copyright© 2020 Springer Nature.

Clioquinol (Fig. 3a), which contains nitrogen and oxygen donor atoms for metal chelation, is also a widely utilized therapeutic framework to target metal ions and Aβ aggregation, as discussed in the previous section.^26,27 Sang and coworkers designed a combinatorial strategy to address Aβ aggregation, metal ion dyshomeostasis, and MAOB activity by incorporating metal-binding donor atoms like clioquinol and Schiff base moieties into a backbone, as depicted in Fig. 9b.³⁰³ Specifically, 3b with 2-hydroxy moiety obtained the lowest IC₅₀ value of 8.4 nM.³⁰³ SAR analysis revealed that an increase in the length of the linker domain or elimination of the hydroxyl group from the aromatic ring dramatically decreased their inhibitory activity.³⁰³ As illustrated in Fig. 9b, 3b prohibited MAOB activity through binding at its active site; notably, the hydroxyl groups of 3b generated hydrogen bonding with E84 and T201, which are essential for the catalytic activity of MAOB. In the absence of hydroxyl groups or additional methyl linkers, the compound no longer maintains its hydrogen bonding network, dramatically weakening binding affinity.³⁰³ Additionally, 3b altered Aβ₄₂ aggregation, reducing fibril growth by 32% and 62% in the presence and absence of Cu(II), respectively. Furthermore, thinner and shorter fibrils were produced in the presence of 3b, supporting the modulatory role of 3b in Aβ₄₂ aggregation.³⁰³ These findings suggest that 3b has potential as a multipotent therapeutic agent confronting MAOB, intracellular ROS, and metal-free and Cu(II)-bound Aβ₄₂.

3.6 Chemical reagents capable of controlling Aβ, AChE, and MAOB

While targeting Aβ, AChE, and MAOB individually has shown promise in addressing specific aspects of AD, the complexity of its pathology necessitates a more integrated approach.^{35,288,298,299} The combinatorial interaction of Aβ, AChE, and MAOB has been shown to induce synaptic dysfunction through synergistic effects, resulting in impaired cholinergic, dopaminergic, and serotonergic neurotransmission.^{35,288,298,299} Therefore, simultaneously tackling all three mechanisms could improve therapeutic outcomes by alleviating symptoms and slowing disease progression. As discussed earlier, compounds should incorporate structural moieties capable of interacting with the CAS and PAS of AChE, the active site of MAOB, and functional groups that effectively disrupt intermolecular hydrogen bonding and hydrophobic interactions of Aβ.

Chromone (Fig. 10a), a heterocyclic compound with a benzopyranone backbone, is a versatile scaffold in drug discovery for various diseases, including AD, due to its antioxidant activity, anti-amyloidogenic properties, and ability to modulate cholinergic and glutaminergic systems.^{308,310–313}Chromone inhibits MAOB activity by forming intermolecular hydrogen bonds between C172 and Y326 through its sp² oxygen atom, a feature unique to MAOB.³¹⁴ Several SAR studies have further explored the chromone backbone to enhance its multi-targeting capabilities, as shown in Fig. 10a.^308,312,313 Kumar and coworkers found that attaching pyrimidine derivatives to the chromone backbone introduces a tertiary nitrogen moiety as a pharmacophore, enabling additional cation–π interaction with W84 in AChE.³⁰⁸Chromone derivatives (Fig. 10a) demonstrated strong inhibitory activity against MAOB and AChE, while chromone-1, featuring a pyrrolidine group at the C4 position, showed the highest potency (IC₅₀ = 2.1 μM for MAOB and 80 nM for AChE).³⁰⁸ In MAOB, the phenyl ring of chromone-1 engages in π–π stacking with Y236, while pyrrolidine and morpholine rings at C4 form electrostatic interactions with P102, F103, and W119, surrounding the FAD cofactor. These interactions are absent in chromone-3, which has a morpholine ring at the C3 position, emphasizing the importance of functional group positioning in designing multi-target-directed molecules.³⁰⁸ All chromone derivatives exhibited similar interactions with AChE, including π–π stacking with F331 and W279 at the PAS and cation–π interactions with W84 at the CAS, underscoring the critical role of the tertiary nitrogen group in AChE inhibition.³⁰⁸ In addition, treatment with chromone compounds restricted the formation of Aβ fibrils and plaques, as evidenced by the loss of aggregate structures in the images obtained by field emission scanning electron microscopy (FE-SEM) (Fig. 10a).³⁰⁸ These findings emphasize the potential of the chromone scaffold in developing multi-target-directed molecules against MAOB, AChE, and Aβ.

Fig. 10 Examples of chemical reagents regulating Aβ, AChE, and MAOB. (a) Structures of chromone and its derivatives, binding of chromone-1 to MAOB, and its impact on AChE activity and Aβ₄₂ aggregation. The inhibitory effect of chromone-1 against the activity of AChE was performed using Ellman's method and represented in a double reciprocal Lineweaver–Burk plot. The morphology of Aβ₄₂ aggregates generated with or without chromone-1 was obtained by FE-SEM. Reproduced with permission from ref. 308. Copyright© 2024 American Chemical Society. Chromone-1, 2-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4H-chromen-4-one; chromone-2, 2-(3-methoxy-4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)-4H-chromen-4-one; chromone-3, 2-(3-(2-morpholinoethoxy)phenyl)-4H-chromen-4-one. (b) Structure of BV-14, its binding mode towards MAOB, and effects on the Aβ₄₂ aggregation and AChE activity. The extent of the inhibition of Aβ₄₂ aggregation was assessed using the ThT assay. The AChE inhibition by BV-14 was measured using Ellman's method and shown in an overlaid Lineweaver–Burk reciprocal plot. BV-14, (Z)-4-(1-phenylprop-1-en-2-yl)-6-(4-(prop-2-yn-1-yloxy)phenyl)pyrimidine. Reproduced with permission from ref. 309. Copyright© 2024 Royal Society of Chemistry. (c) Structure of CF-4, its binding to MAOB (PDB 1GOS¹¹³), and impact on the production of Aβ₄₂ aggregates. The morphology of the resultant Aβ₄₂ species was analyzed by TEM. CF-4, (E)-4-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-3-oxoprop-1-en-1-yl)-2-methoxyphenyl ethyl(methyl)carbamate. Reproduced with permission from ref. 310. Copyright© 2020 Elsevier.

In a separate study, Kumar and coworkers designed phenylstyrylpyrimidine derivatives containing O-propargyl and amidine moieties, a combination of previously known pharmacophores, to evaluate their multi-target potential.^309,315,316 The study demonstrated the inhibition of MAOB activity, and a SAR analysis revealed that BV-14 (Fig. 10b), featuring a formamidine group, outperformed other functional derivatives with guanidine, acetamidine, or benzamidine groups, attaining an IC₅₀ of 7.3 μM.³⁰⁹BV-14 suppressed MAOB activity by binding to the active site, with its pyrimidine ring and propargyl group engaging with the Y326 residue and the FAD cofactor of MAOB through hydrophobic interactions, including π–π stacking, and additional hydrogen bonds (not shown in the figure).³⁰⁹ In addition, BV-14 showed the inhibitory effect on AChE, possessing IC₅₀ of 7.3 μM (Fig. 10b), which was achieved by the hydrogen bonding with F288, a residue from the PAS.³⁰⁹ To assess the broader impact of BV-14 on AD-related factors, researchers also evaluated its influence on prohibiting ROS generation and Aβ aggregation. Although detailed insights were not provided, BV-14 lowered ROS production by 48%, as depicted in Fig. 10b. Additionally, BV-14 prevented Aβ₄₂ aggregation, reducing fibril growth by 22%. These findings suggest that BV-14 has potential as a multipotent therapeutic agent targeting MAOB, intracellular ROS, and Aβ₄₂.³⁰⁹

Natural products have also been explored for their potential to modulate Aβ aggregation, along with AChE and MAOB activity. Ferulic acid, a widely distributed plant constituent first isolated from Ferula foetida, has been investigated as a therapeutic agent for AD in this manner.^317,318 Chen and coworkers designed O-carbamoyl ferulamide derivatives by combining ferulic acid with a carbamate fragment, a pharmacophore found in rivastigmine (Fig. 4a), to create compounds with multi-targeting potency against MAOB, AChE, and Aβ.^310,319 Among these derivatives, CF-4 (Fig. 10c) emerged as a promising and selective MAOB inhibitor with an IC₅₀ of 5.3 μM. CF-4's carbamoyl group interacted with the FAD cofactor, forming intermolecular hydrogen bonds and π–π interactions with the Y60 residue. The carbonyl group of the ferulic acid backbone generated hydrogen bonds with T201 and Q206, emphasizing the importance of the ferulic acid moiety in binding to the active site of MAOB.³¹⁰ Furthermore, CF-4 not only interacted with the active site of MAOB but also exhibited inhibitory effects on AChE. The benzene ring of the 1,2,3,4-tetrahydroisoquinoline and the ferulic acid created π–π interactions with key amino acid residues in AChE, enabling CF-4 to simultaneously associate with both the CAS and the PAS, leading to effective suppression of AChE.³¹⁰ Additionally, CF-4 prohibited Aβ₄₂ aggregation by up to 58%,³¹⁰ with a significant reduction in bulk Aβ₄₂ aggregates (Fig. 10c). Notably, CF-4 also showed self-mediated disaggregation of Aβ₄₂ by 43%, demonstrating its dual capacity to alter and disaggregate Aβ₄₂ aggregates.³¹⁰CF-4's ability to cross the BBB permeability and its efficacy in improving cognitive defects induced by scopolamine further validated its potential for clinical use.³¹⁰ These findings on CF-4, with those of chromone derivatives and BV-14, underscore the importance of targeting AChE, MAOB, and Aβ in AD therapy. This comprehensive approach, which combines existing therapeutics to address the complex pathology of AD, offers a more efficient strategy for disease treatment.

4. Conclusions

With the rising prevalence of AD, there is an increasing need for effective therapeutic strategies. The limitations of current single-target-directed treatments highlight the necessity for more comprehensive approaches that address the multifactorial nature of AD. This review illustrates several pathological features in AD, particularly Aβ aggregation, metal ion dysregulation, and enzymes' dysfunction. While Aβ has long been considered a primary therapeutic target due to its role in disrupting cellular functions, inducing inflammation, and impairing synaptic activity, treatments focused solely on Aβ have often demonstrated limited success. Although recent drug approvals aiming at Aβ underscore its continued relevance as a therapeutic biomarker, it is essential to concurrently engage additional neurotoxic factors contributing to AD progression to effectively combat the disease. The complex interplay of metal ion dyshomeostasis, metalloenzyme dysregulation, impaired neurotransmission, and elevated oxidative stress further complicates the pathology of AD and constrains the success of isolated interventions.

As such, the development of multi-target-directed molecules capable of modulating multiple pathogenic pathways offers a promising strategy to enhance therapeutic efficacy. Future research should prioritize the refinement and optimization of MTDLs to address the interconnected mechanisms associated with AD. A more systematic therapeutic approach that simultaneously tackles Aβ aggregation, metal ion dyshomeostasis, enzyme dysregulation, and neurotransmitter imbalance holds the potential to overcome the limitations of current single-target-directed treatments. Alongside the multi-target-directed molecules primarily focusing on Aβ, extensive research has also been conducted on therapeutic combinations not covered in this review, which underscores their significance and potential in advancing AD treatment strategies. Furthermore, beyond the pathogenic targets discussed in this review, the development of MTDLs aimed at neuroinflammation, mitochondrial dysfunction, and neuronal cell death could provide new insights into their interconnected roles in AD pathogenesis and therapeutic intervention. A deeper understanding of these processes and their interplay with previously discussed factors could guide the design of MTDLs capable of effectively confronting the complex, multifaceted nature of AD. Expanding the scope of therapeutic targets, as discussed in this review, can serve as a foundation for future research, driving the development of disease-modifying treatments that efficiently address the complexity of AD.

Data availability

This manuscript is a review article; therefore, no new data were generated or analyzed in this study, and a data availability statement is not applicable.

Author contributions

All authors contributed to the conceptualization of this review. Jeasang Yoo, Jimin Lee, Byeongha Ahn prepared the draft, which was revised by Professors Mi Hee Lim and Jiyeon Han.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by grants of the National Research Foundation of Korea funded by the Korean Government {Creative Research Initiative [RS-2022-NR070709 (M. H. L.)]; Global Science Research Center Program [RS-2024-00411134 (M. H. L.)]; RS-2024-00353015 (J. H.)}.

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Footnote

† These authors contributed equally to this work.

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