Polyoxometalates: metallodrug agents for combating amyloid aggregation

ABSTRACT Alzheimer's disease (AD) is a devastating neurodegenerative disease that affects ∼50 million people globally. The accumulation of amyloid-β (Aβ) plaques, a predominant pathological feature of AD, plays a crucial role in AD pathogenesis. In this respect, Aβ has been regarded as a highly promising therapeutic target for AD treatment. Polyoxometalates (POMs) are a novel class of metallodrugs being developed as modulators of Aβ aggregation, owing to their negative charge, polarity, and three-dimensional structure. Unlike traditional discrete inorganic complexes, POMs contain tens to hundreds of metal atoms, showcasing remarkable tunability and diversity in nuclearities, sizes, and shapes. The easily adjustable and structurally variable nature of POMs allows for their favorable interactions with Aβ. This mini-review presents a balanced overview of recent progress in using POMs to mitigate amyloidosis. Clear correlations between anti-amyloid activities and structural features of POMs are also elaborated in detail. Finally, we discuss the current challenges and future prospects of POMs in combating AD.


INTRODUCTION
Alzheimer's disease (AD), a neurodegenerative disorder associated with aging, affects ∼50 mi l lion people and begets an ongoing socio-economic burden globally [1 ].The dominating pathological hallmarks of AD are extracellular amyloid-β (A β) plaques and intracellular neurofibri l lary tangles [2 ,3 ].Although the detailed etiology of AD remains unclear, the mainstream belief now is that the widespread accumulation of A β peptide in the brain is an initial and pivotal event in the progression of AD, further eliciting a series of detrimental cascades, including tau pathology, chronic inflammation, and cognitive malfunction [4 -7 ].Recently, three anti-amyloid monoclonal antibodies, aducanumab, lecanemab, and donanemab, have been approved for AD therapy by the U.S. Food and Drug Administration (FDA).These antibodies can clear A β plaques and postpone cognitive decline in early-stage AD patients, validating the therapeutic strategy of targeting A β [8 ].Therefore, modulation of A β aggregation is perceived as a promising strategy in the fight against AD.Numerous metal compounds have been developed as metallodrug agents targeting various diseases, including diabetes, cardiovascular diseases, and cancer [9 -12 ].Lately, metallodrugs have gained considerable interest as potential modulators of A β aggregation, due to their remarkable physicochemical properties such as diverse coordination geometries and multiple metal center oxidation states [13 -20 ].These metallodrugs are capable of disrupting A β aggregation and alleviating A β-related neurotoxicity through both direct and indirect interactions with A β, including electrostatic attraction, coordination, π -π stacking, oxidation, and hydrolysis.Polyoxometalates (POMs), a burgeoning class of metallodrugs, are often described as discrete clusters of early transition metal oxides, with the central metal ions typically in their highest oxidation state [21 -24 ].POMs can be divided into two primary groups: iso-POMs [M x O y ] n − and hetero-POMs [X x M y O z ] n − (where M = V, Mo, W, Nb, Ta; X = B, As, Si, P, Ge, etc.) [25 ,26 ].Both iso-POMs and hetero-POMs generally consist of octahedral [MO 6 ] synthons interconnected by bridging oxo atoms.While numerous POM structures have been identified to date, most studied POMs fall into one of the four prevalent POM archetypes (Anderson-, Keggin-, Wells-Dawson-, and Lindqvist-type structures) [25 ,27 ], as depicted in Fig. 1 .POMs hold significant advantages over traditional discrete inorganic clusters, as they can be easily derived from cost-effective inorganic salts and provide a broad spectrum of structures with diverse elemental compositions, geometries, and solution stability [28 -33 ].Crucially, their associated chemical properties, such as redox potential, acidit y, polarit y, and surface charge distribution, can be precisely tailored to specific needs by adjusting factors like composition, size, shape, and counterion [34 ,35 ].These outstanding features gift POMs with multifarious bioactivities in the figh t agains t ca nce r, viruses, diabetes, and bacteria [27 ,31 ,33 ,36 -39 ].The pharmacological and biological attributes of POMs are largely ascribed to their interactions with proteins [33 ,40 ,41 ].Exhilaratingly, several studies have examined the interactions between POMs and proteins [41 -43 ].As revealed by various techniques such as X-ray crystallography, computational modeling, and isothermal titration calorimetry, POMs typically form multiple types of interactions with proteins, including electrostatic, hydrogen bonding, van der Waals, and coordination interactions (Fig. 2 ) [41 -43 ].Electrostatic attractions are the dominant forces in POM-protein interactions, due to the polyanionic and oxygen-rich nature of POMs.Specifically, POMs tend to preferentially interact with protein regions containing positively charged functional groups.Alongside electrostatic interactions, noncovalent binding of POMs with proteins is predominantly governed by hydrogen bonds.Instead of outlining the progress on specific protein interactions with POMs, Parac-Vogt et al. recently overviewed the general principle on POM-protein interactions, guiding the field development [43 ].Such direct interactions of POMs with proteins enlightened us to explore their effects on A β folding and conformation.Our first preliminary works focused on the modulation effect of POMs toward A β aggregation [44 ].Unsurprisingly, the screening results indicated that these typical POM compounds, including Wells-Dawson and Keggin structures, bound strongly to A β and hindered A β aggregation [44 ].The nascent POMs-based strategy for modulating A β aggregation may shed light toward the design and screening of cost-effective metallodrugs to treat devastating AD.
In this review, we delineate and classify the advances of POMs based on their means of intervening with the aggregation of A β and site-directed modification of A β (Fig. 3 ).The discussion mainly focuses on their design, working mechanisms toward the aggregation of A β, and corresponding applications in AD treatment.At the end, the scientific opportunities and challenges for future advancement in the field are also discussed.

Anti-amyloid activity of POMs
With the in-depth understanding of POM-protein interactions, our group demonstrated that POMs could efficiently block A β 40 conformational changes and redirect A β 40 aggregation into off-pathway, unstructured aggregates (Fig. 4 ) [44 ].The inhibition activity and selectivity of POMs resulted from size-specific electrostatic interactions between POMs and the cationic cluster His13-Lys16 of A β, which primarily depends on the size and charge of POMs.fibri l lization [45 ].In addition, Liu's group found that the spherical shape of POMs exhibited strong inhibitory effects against A β aggregation [46 ].Intriguingly, they also prevented Cu 2 + -or Zn 2 +induced A β aggregation and blocked A β-mediated neurotoxicity.
Previous studies have shown that the distinctive interaction between POMs and A β largely relies on electrostatic attractions.Although the POMs-A β electrostatic interactions are robust, the binding affinity of POMs to A β could sti l l be improved.Notably, this cationic His13-Lys16 (HHQK) cluster in A β comprises two adjacent histidine amino acids, which are able to chelate transition metal ions.Consequently, our group constructed a variety of transition-metal (Mn, Cu, Fe, Co, Ni)-substituted Wells-Dawson POMs (POMds) [47 ].Engineering of a high-throughput screening approach based on the cyan fluorescent protein (CFP)-fusion expression system, we demonstrated that POMds exhibited stronger A β binding affinity and superior A β inhibitory effect compared with the parent Wells-Dawson POMs (Fig. 5 A and B).This was ascribed to the robust coordinating interactions between POMds and His13/His14 (Fig. 5 C).In addition, POMds could reduce A β-heme peroxidase-like activity.More intriguingly, increased accumulation of POMds in the brain was observed, peaking at 10 minutes after injection, which suggested their potential ability to pass through the blood-brain barrier (BBB).Moreover, the level of POMds in the brain began to decrease after 10 minutes post-injection and returned to initial levels after 48 hours under the experimental conditions.These results indicated that POMds could be cleared from the brain over time, avoiding long-term toxicity.Although POMds could cross the BBB, just a small fraction of the injected dose actually reached brain tissues due to their suboptimal BBB permeability and selectivity.Furthermore, Hureau's group found that the lacunary POMs with Keggin structure removed Cu 2 + ions bound to A β, stopped A β-Cu 2 + complexinduced reactive oxygen species (ROS) production, and modified A β aggregation (Fig. 5 D) [48 ].

Anti-amyloid activity of POMs-organic hybrids
The POMs-A β interaction primarily relies on sizedependent electrostatic interactions.Therefore, pristine POMs sti l l need improving their selectivity when binding to A β.The covalent/coordination modification of pristine POMs with bioactive ligands provides an opportunity to develop POMsorganic hybrids, which can increase selectivity and reduce toxicity [49 ,50 ].Bioactive ligands refer to a kind of molecule that can specifically bind to a target biomolecule such as protein or receptor, and modulate its biological function or activity [51 ].Currently, several bioactive ligands (e.g.small molecules, peptides, and antibodies) have been incorporated into POM hybrids to acheive safe and selective inhibition of A β aggregation.For example, Ma et al. employed an A β-targeting organometallic group CoL 2 + [L = 2-(1Hpyrazol-3-yl)pyridine] for the hybridization with ε-Keggin (HAsMo 12 O 40 ) 8 − unit to develop a newly modified POM with excellent targeting ability toward A β (Fig. 6 A and B) [52 ].The organocobalt-substituted Keggin POM O 40 ] (abbreviated as CAM) selectively interacted with A β and disaggregated self-aggregated or metal-induced A β fibrils (Fig. 6 C).This effect was attributed to the matching size of CAM and the cavity of A β aggregates, and the hydrogen-bonding interactions of CAM with amino residues in the cav ity.A s a result, CAM suppressed the production of ROS and decreased the synaptic toxicity of A β aggregates.Importantly, the well-designed CAM was lipophilic and penetrated the BBB.These results highlight the power of combining POMs with known A β-targeting ligands for more specific interactions.
Zhao et al. synthesized an organoplatinumsubstituted Keggin POM for blocking A β aggregation (Fig. 6 D) [53 ].In comparison with the unmodified POM, the interaction of the Ptsubstituted POM with A β was greatly improved and more specific, which was attributed to the synergistic effect of Pt II ions with amino acid residues in A β.These multiple strong interactions endowed Pt-substituted POM with a remarkable inhibitory effect on A β fibri l lation (IC 50 = 0.62 μm).In cell-based experiments, the Pt-substituted POM significantly reduced A β aggregation-mediated neurotoxicity.Moreover, in vivo experiments verified the Pt-substituted POM decreased A β deposition and alleviated cognitive impairments in AD model mice without apparent systemic toxicity (Fig. 6 E).These results emphasize the significance of structural modification of POMs to achieve enhanced and selective POMs-A β interactions.
Chirality is the inherent characteristic of aggregated A β proteins, which can be classified into 4 levels (Fig. 7 A) [54 ].The first level is called 'configurational chirality' , denoting the asymmetric arrangement of an atom with a set of ligands.The second level is known as 'conformational chirality' , denoting the different helical conformation of A β.The third level is 'structural chirality' , denoting the phase structure of A β fibrils.The fourth level is 'object chirality' , which usually comes from the accumulation of helical single domains to form mesoscopic or macroscopic chiral objects.Many studies show that the chiral variations of amino acids are able to affect A β specificity and induce A β isomerization and epimerization, which are closely associated with AD dysfunction [55 ,56 ].Therefore, the chirality strategy has garnered widespread attention in the design of amyloid inhibitors.Recently, our group prepared a range of chiral Anderson POMs modified with amino acids, including hydrophobic D-/L-Phe and Leu amino acids, negatively charged D-/L-Glu amino acids, and positively charged D-/L-His amino acids, for chirality-selected inhibition of A β fibri l lation (Fig. 7 B) [54 ].According to fluorescence titration, isothermal titration calorimetry, circular dichroism, and ThT fluorescence assays, D-/L-Phe-modified chiral POMs exhibited higher binding affinity to A β and stronger inhibition effect against A β aggregation, compared to other D-/L-amino acid modified chiral POMs.Moreover, D-Phe-modified chiral POMs exerted a better suppression effect than the enantiomer L-Phe-modified chiral POMs because the Phe4, Ser8, His13, and Phe19 amino acid residues of A β preferred closing to D-Phe on the surface of POMs (Fig. 7 C).More intriguingly, the chiral POMs crossed the BBB and prolonged the Caenorhabditis elegans CL2006 strain lifespan.Overall, these findings highlight the potential of enhanced POM-A β interactions through chiral ligand functionalization.

Anti-amyloid activity of POMs-based nanocomposites
POMs-A β interactions can also be tuned through supramolecular self-assembly to form POM-peptide hybrid nanocomposites.Many peptides/peptide mimetics possess strong anti-amyloid properties and are therefore an excellent option for hybridization with POMs to construct novel nanocomposites with improved biological activity [57 -59 ]  , with the wellknown β-sheet breaker QKLVFF to self-assemble in nanospheres (abbreviated as POM@P) (Fig. 7 D) [60 ].The two-in-one POM@P nanocomposites exhibited both enhanced A β binding specificity and suppression of A β aggregation.Furthermore, by incorporating a probe molecule, Congo red, into the POM@P nanospheres, the nanocomposites could be utilized to detect the inhibitory effect of POM@P on A β aggregation in real time.
The AuNPs@POMD-pep displayed synergistic effects on preventing A β aggregation and disassociating the formed A β fibrils.Moreover, through the use of AuNPs as BBB-transport vectors, the AuNPs@POMD-pep effectively crossed the BBB.

Modulate A β aggregation through covalent modification
Most POMs-based inhibitors bind to A β through non-covalent interactions, which are susceptible to the surrounding environment and inherently weak.However, covalent interactions are not influenced by fluctuations in the surrounding environment and are chemically stable.It is well established that proteins undergo a variety of post-translational modifications (PTMs), such as covalent conjugations of glycans or phosphate groups to amino-acid side chains.Such covalent modifications can significantly change protein structures and activities.A β peptides have diverse PTMs that variously modulate both the aggregation states and bioactivities of A β [63 ].Unfortunately, it is challenging to control PTM manually because of diverse subcellular localizations and intricate cellular signaling pathways.Thus, it is demanding to develop convenient and efficient chemical PTM agents.
For the first time, our group rationally designed a Wells-Dawson POMs-based PTM agent for site-directed chemical modification of A β (Fig. 8 ) [64 ].Following the functionalization of POMs with thiazolidinethione (TZ), the resulting POMD-TZ served as a chemical PTM agent, covalently modifying A β at Lys16 residues (Fig. 8 A).POMD-TZ exhibited a much stronger inhibitory effect on A β aggregation than the unmodified POMD.Moreover, in vitro biophysical and biochemical assays indicated that POMD-TZ site-specifically modified A β, modulated aggregation and mitigated A β aggregationcaused cytotoxicity.These results highlight the potential of covalent modification to construct new hybrid POMs with tailored bio-functionalities.
Next, we improved the therapeutic effect of chemical PTM agents in two ways: (i) to increase A β targeting ability, we added a TZ-modified aspartic acid (D) at the N-terminal of the A β targeting peptide LVFFA to synthesize POMD-Tar-TZ.(ii) To enable POM-TZ to differentiate between A β monomers and oligomers, we introduced a fluorescence resonance energy transfer (FRET) probe to produce POMD-Tar-TZ-FRET (Fig. 8 B).The FRET between the naphthalimide-based fluorescent probe and rhodamine increased the sensitivity of A β oligomer detection.As a result, POMD-Tar-TZ-FRET not only covalently modified A β but also selectively detected and visualized A β oligomers.

POMS AS PHOTOSENSITIZERS FOR PHOTO-INDUCED INHIBITION OF A β AGGREGATION
Because of its low invasiveness and high spatiotemporal controllability, phototherapy has been extensively explored for the treatment of various localized diseases involving pancreatic, prostate, and breast cancers [65 ,66 ].However, phototherapy has been seldom considered for the application in neurodegenerative diseases [67 ,68 ].POMs, which generally exhibit reversible multi-electron redox transitions, are very promising photocatalysts for diverse applications such as water splitting, pollutants degradation, and cancer therapy [34 ,69 ,70 ] Ma et al. also prepared a series of redox-activated POMs with Keggin structure for photothermal treatment of AD (Fig. 9 B) [72 ].Reduced POMs (rPOMs) were nicely embedded in the mesoporous silica nanoparticles (MSNs).Then, the resulting rPOMs@MSNs were incorporated into thermoresponsive polymers for preventing the leakage of rPOMs.On one hand, under near-infrared (NIR) light irradiation, rPOMs could produce local hyperthermia to destroy mature A β fibrils.On the other hand, the external polymers were melted by the local heat responses, thereby releasing rPOMs.The released rPOMs not only hindered A β aggregation but also scavenged superfluous ROS.Intriguingly, among these used rPOMs, rPOM with Wells-Dawson structure showed a much better behaviour.

POMS AS ARTIFICIAL ENZYMES/NANOZYMES FOR DEGRADING A β
A growing body of research reveals that the accumulation of brain amyloid plaques is not only correlated with A β generation, but also dependent on A β degradation.Proteolysis of A β, a very promising method to lower brain A β levels and to mitigate A β-mediated neurotoxicity, has gained much attention in recent years [73 ].Currently, various natural enzymes, including insulin-degrading enzyme, endothelin converting enzyme, and neprilysin, have been developed for efficient A β hydrolysis [74 ,75 ].However, the intrinsic limitations of natural enzymes, such as poor stability, high cost, and cumbersome purification, severely restrict their practical applications.In these contexts, A β-degrading artificial enzymes have emerged as a potential alternative to natural enzymes [76 -78 ].Recently, Gao et al. designed and synthesized a POMs-based artificial enzyme (denoted as AuNPs@POMD-8pep) for multi-faceted treatment of A β aggregates (Fig. 10 A  and B) [79 ].The AuNPs@POM-8pep was composed of three ingredients: Wells-Dawson POM (POMD) that attacked the peptide bond, AuNPs that promoted electron transfer and increased the hydrolysis rate, and octa-peptide (N-Cys-His-Sar-His-Sar-His-Sar-His) (Fig. 10 A).The resulting AuNPs@POMD-8pep possessed both high protease-like activity and superoxide dismutase (SOD)-like activity, which depleted A β aggregates and scavenged A β/Cu-induced ROS (Fig. 10 B).Intriguingly, AuNPs@POMD-8pep could chelate Cu(II) ions and cut off Cu(II)-expedited A β aggregation.The AuNP@POMD-8pep was subsequently covalently functionalized with an A β-targeted peptide (CLPFFD), which could stem misdirected or undesirable proteolytic hydrolysis reactions.More importantly, the AuNPs@POMD-8pep efficiently crossed the BBB.
Guan et al. also constructed a series of Ceria/POMs nanohybrids (CeONP@POMs) with both robust protease-like activity for degrading A β peptides and high SOD-like activity for scavenging intracellular ROS (Fig. 10 C) [80 ].Among them, CeONP@POMD with Wells-Dawson type performed the highest hydrolysis activity toward A β.The CeONP@POMK with Keggin type showed moderate hydrolytic activity.The CeONP@POMA with Anderson type possessed the lowest activity.The CeONP@POMD not only facilitated PC12 cell proliferation, but also diminished BV2 microglial cell activation caused by A β.More intriguingly, CeONP@POM could cross the BBB and possess good biocompatibility.

CHALLENGES AND OUTLOOK
POMs, a promising class of metallodrugs, are capable of effectively inhibiting A β aggregation.POMs are known to bind to A β mainly through sizedependent electrostatic interactions with positively charged His13-Lys16 (HHQK) regions of A β [44 ].It is well established that the cationic cluster HHQK plays key roles in both A β oligomerization and fibril propagation.Thus, this binding could efficiently block A β conformational changes and shift the equilibrium away from fibri l lization.There are numerous factors known to affect POM-A β interactions.Owing to the primarily electrostatic nature of the interactions, the POMs' charge plays a pivotal role on its inhibition ability.Besides, the size of POMs also influences POM-A β interactions.For example, relatively large Wells-Dawson POMs exhibit a stronger inhibition effect than Keggin and Anderson types.Last but not least, the ability of POMs to gain access to the binding pocket in A β aggregates based on their shape can affect POM-A β interactions [52 ].
Although the inhibitory effect of POMs is quite promising, purely inorganic POMs exhibit limited A β selectivity.The functionalization of POMs offers a direct and highly controllable approach to modify both the structure and bioactivity of POMs.Enhanced POM-A β interaction can be achieved by (i) covalent/coordination modification with bioactive molecules and metal ions, or (ii) constructing POM-based nanocomposites.Collectively, the emergence of hybrid POMs offers a promising strategy for combining POMs with targeting moieties to achieve novel POM-based inhibitors with reduced toxicity and enhanced specificity.
POMs, featuring adjustable compositions and varied structures, have been used as electro/photocatalysts because of their unparalleled benefits, including quasi-semiconductor traits, exceptional redox and solution stability [3 0 , 3 4 ,69 ,70 ].Moreover, POMs have gained considerable interest due to their attractiveness as building blocks for the construction of multi-functional nanocomposites [22 ,26 ].In addition to inhibiting A β aggregation by direct binding to the HHKQ region, POM-based nanocomposites are known to act as photosensitizers for photo-induced inhibition of A β aggregation [71 ].Moreover, POM-based nanocomposites with highly Lewis acidic metal centers exhibit excellent hydrolysis activity toward A β peptides [80 ].To tackle poly-pathological features in AD, the rational design of multi-functional materials by integrating two or more therapeutic moieties into one nanocomposite has gained particular attention [4 ].Taking advantage of the flexible structural modifiability of POM, designing multi-functional POM-based therapeutic agents is a promising strategy for AD therapy.
Although meticulously designed POM structures yield desired properties, the intricacy of POM systems presents considerable obstacles in comprehending POM-A β interactions under physiological conditions.This is because, in general, the POM-A β interactions involve multiple weak non-covalent interactions, including electrostatic interaction, hydrogen bond, and coordination interaction.Thus, POM-A β interactions are highly susceptible to environmental alterations, such as pH, temperature, and ionic strength [21 ,23 ,24 ,29 -33 ,39 ,40 ,42 ].In addition, the relatively low stability and suboptimal biocompatibility of POMs in physiological environments wi l l hinder their application in AD therapy [38 ,81 ].Moreover, the surface features of POMs limit their infiltration into cells, leading to potential cytotoxicity [40 ].Hence, predicting and analyzing POM-protein interactions under physiological and pathological conditions, as well as exploring the mechanism of POM toxicity actions, are crucial challengs that need to be addressed, especially for the development of POM-based therapeutic agents.
Despite these extensive progresses, POMs as anti-amyloid agents are sti l l in the infancy of development and there is a long way to go before clinical applications.Many issues sti l l exist, such as insignificant BBB penetration, high cytotoxicity, and unclear biological and pharmacokinetic properties.Much more work is undoubtedly necessary to overcome those challenges and pave the way for POMs as the next generation of anti-AD metallodrugs under the interdisciplinary cooperation of researchers, such as structral biologists, medicinal chemists, biochemists, and neurologists.We are only at the dawn of POMs targeted toward the inhibition of A β aggregation, and expect to shed light on the developments of AD treatment in the near future.

Figure 2 .
Figure 2.Overview of the common POM-protein interactions, including electrostatic interaction between POMs and proteins containing positively charged functional groups, hydrogen bonding between POMs and proteins containing hydroxyl functional groups, van der Waals interaction between POMs and proteins containing methyl functional groups, and coordinative binding between substituted metal ions in POMs and oxygen or nitrogen atoms in proteins.

Figure 3 .
Figure 3. Schematic representation of anti-A β activity of POMs.
. Inspired by the unique photocatalytic activities of POMs, Li et al. found that Wells-Dowson POMs K 8 [P 2 CoW 17 O 61 ] displayed enhanced inhibition effects against A β self-assembly under UV irradiation (Fig. 9 A), which was ascribed to the photodegradation of A β by the singlet oxygen ( 1 O 2 ) released from the photocatalyst K 8 [P 2 CoW 17 O 61 ] [71 ].The work presented here took a major step toward the development of AD phototherapy.

Figure 9 .
Figure 9. (A) Schematic illustration of POMs used to modulate A β aggregation upon UV irradiation.(B) Schematic drawing of redox-activated POMs for photothermal treatment of AD.Adapted with permission from ref. [71 ]; Royal Society of Chemistry; ref. [72 ], Wiley.
. Inspired by this phenomenon, Li et al. combined a Wells-Dawson POM K 8 [P 2 CoW 17 O 61