Small molecules to target tau amyloid aggregation

Zoe Manglano-Artuñedo,Samuel Peña-Díaz ,Salvador Ventura

Protein aggregation has been linked with many neurodegenerative diseases,such as Alzheimer’s disease (AD) or Parkinson’s disease.AD belongs to a group of heterogeneous and incurable neurodegenerative disorders collectively known as tauopathies.They comprise frontotemporal dementia,Pick’s disease,or corticobasal degeneration,among others.The symptomatology varies with the specific tau protein variant involved and the affected brain region or cell type.However,they share a common neuropathological hallmark -the formation of proteinaceous deposits named neurofibrillary tangles.Neurofibrillary tangles,primarily composed of aggregated tau(Zhang et al.,2022),disrupt normal neuronal functions,leading to cell death and cognitive decline.

Tau is an intrinsically disordered protein that contributes to microtubule stabilization and plays an important role in axonal transport.The tau protein sequence can be dissected into four domains: i) the amino-terminal domain that contains up to two amino acid inserts (N1 and N2);ii) a proline-rich segment;iii) the microtubulebinding domain,a positively-charged region that encloses 3/4 imperfect repeats (R1,R2,R3,and R4),and delineates two aggregation-prone hexapeptides (at R2 and R3);and iv) the carboxyterminal domain.Remarkably,alternative splicing gives rise to six different isoforms that vary depending on the presence or absence of R2 (K18/K19 fragment,respectively) and of the N inserts(Figure 1A).Additionally,tau also undergoes numerous post-translational modifications,including acetylation,ubiquitination,or phosphorylation,which has been proposed to play a key role in tau function and aggregation.In pathological conditions,hyperphosphorylation reduces tau-microtubule affinity and promotes aggregation.Tau aggregation is a multifaceted process that involves the generation of various species.The self-assembly of phosphorylated tau monomers initiates the formation of oligomeric species that are often enriched in β-sheet architecture and expose hydrophobic patches to the solvent.These arrangements are thought to be the most cytotoxic structures in amyloidosis.Over time,the oligomeric species evolve into protofibrillar assemblies,which ultimately mature into highly structured fibrils.Noticeably,these fibrils can induce the deposition of soluble tau in healthy neurons,leading to the propagation of pathological amyloids throughout the brain (Zhang et al.,2022).

Figure 1｜Overview of tau sequence and anti-aggregation moieties.

In the quest to halt or slow down the progression of tauopathies,inhibiting tau aggregation has emerged as a promising approach.Multiple strategies have been investigated: i) regulating tau hyperphosphorylation,ii) downregulating tau expression,iii) promoting clearance of phosphorylated tau,and iv) developing modulators of tau amyloid aggregation (Zhang et al.,2022),including antibodies,peptides or small molecules.In this scenario,small molecules are often preferred due to their enhanced ability to penetrate the blood-brain barrier compared to protein-based interventions.For these compounds,a detailed characterization of their inhibitory mechanism,the targeted species(oligomers,protofibrils,and/or fibrils),and a validation of theirin vivoeffect are imperative before they can be evolved into effective drugs for clinical use.

The disordered nature of tau has posed challenges for drug development using structure-based approaches.Methodologies such as highthroughput screening (HTS) or drug repurposing,have emerged as alternatives to identify molecules with inhibitory properties against tau aggregation.Over the years,both strategies have rendered a variety of modulators targeting aberrant tau polymerization (Figure 1B).

In recent years,HTS protocols using amyloid dyes like thioflavin-T or -S have been exploited to discover fibrillization inhibitors of tau truncated variants,which exhibit accelerated aggregation kinetics.In this way,the screening of～51,000 compounds against K18 aggregation led to the discovery of 113F08 and 330B06,which contain a 2,3-di(furan2-yl)-quinoxaline structure,as concentration-dependent modulators of K18 aggregation.In another strategy,the development of a ligand-based computational screening,based on pharmacophore models,identified the phenylthiazol-hydrazide scaffold as a source of inhibitors of K19 aggregation.Another HTS study analyzed the activity of different molecules against K19 heparin-induced aggregation.Compounds like B4D5 or B4D3 significantly decreased the formation of amyloids and even disrupted preformed fibrils (PFFs).Importantly,cell models based on the K18 variant suggested a protective activity against aggregate-mediated toxicity(Longhena et al.,2018).

Drug repositioning has emerged as an alternative to HTS,as it reduces the risk of failure,the time necessary to reach the market,and the cost.For instance,tolcapone and entacapone,two catechol-O-methyltransferase inhibitors prescribed for Parkinson’s disease treatment,exhibited anti-aggregating properties against tau hexapeptides fibrillization.Notably,both drugs share a nitrocatechol scaffold that is believed to underlie the inhibitory activity,opening an avenue for developing nitrocatechol derivatives as tau aggregation inhibitors (Mohamed et al.,2013).Tolcapone has been proposed as a drug candidate for AD due to its superior ability to penetrate the blood-brain barrier compared to entacapone.

Tau aggregation is driven by the two hexapeptide motifs located at R2 and R3 repeats.However,evidence suggests that two neighboring cysteines (C291 and C322) may play an important role in promoting aggregation by stablishing intermolecular disulphide bonds.This discovery prompted a rationale for the development of novel inhibitors.A thiol-reactive rosamine derivative molecule,TR-2,has been reported to exhibit potent activity against K18 fibrillization by binding to and blocking cysteine residues,thus hindering the pathological tau aggregation (Gerson et al.,2014).The C291/C322 reactivity was also employed to develop a strategy to identify covalent inhibitors of K18 fibrillization.As a result,vinylsulfone-derivates were found to interfere with the aggregation process.These findings suggest that the covalent binding of molecules to cysteine residues near the hexapeptide-amyloid motifs can modulate tau aggregation (Petri et al.,2022).

The combination of drug repositioning and rational design resulted in the repurposing of two hybrid molecules,naphtoquinone-tryptophan and naphtoquinone-dopamine as tau aggregation inhibitors.Both molecules were designed based on the critical role of aromatic residues in stabilizing amyloid fibril structures.The quinone skeleton was selected for its reported potential to inhibit the self-assembly of different amyloid proteins,and tryptophan/dopamine molecules were expected to bind the aromatic residues,interfering with the aromatic π-π interactions that sustain the amyloid core.Naphtoquinonetryptophan inhibited the aggregation of one of the tau hexapeptides and ameliorated tauopathyrelated phenotype in an animal model expressing human tau.In contrast,naphtoquinone-dopamine inhibited the aggregation and disassembled PFFs of both hexapeptide motifs and Tau2N4R(Dominguez-Meijide et al.,2020).Interestingly,these molecules have been found to efficiently cross the blood-brain barrier,offering a potential therapeutic application in AD.

K18/K19 variants have been used to pre-screen the inhibitory activity of anti-aggregational compounds,subsequently assayed on top of tau natural variants.Certain modulators displayed a polyphenol-scaffold,including PE859,which prevented K18/K19 and 2N4R aggregation and showed efficacy in mouse models,and oleocanthal,which prevented K18 and Tau2N4R aggregation by covalently binding to lysine residues on the hexapeptide repeat via its aldehyde groups(Dhouafli et al.,2018;Longhena et al.,2018;Wang et al.,2021).Similarly,anthraquinones such as emodin or asperbenzaldehyde,which share a tricyclic aromatic ring scaffold,were identified as tau aggregation inhibitors in an HTS using K19 and further validated in 0N3R and 0N4R variants.Interestingly,anthraquinones also inhibited K18∆K280 aggregation,which does not require heparin for fibrillization,demonstrating that the inhibitory activity did not rely on compoundheparin interactions (Gerson et al.,2014;Longhena et al.,2018).Another HTS exploiting K18P301L aggregation led to the discovery of aminothienopyridazynes,which exhibited antiaggregating features without interfering with tau-mediated microtubules assembly and an inhibitory effect in 2N4R (Gerson et al.,2014).Remarkably,despite effectiveness,most of these molecules were the worst inhibitors of naturaloccurring variants aggregation compared to K18/K19,suggesting potential variations in the mode of action for truncated and naturally occurring isoforms.

Not surprisingly,the exclusive use of natural isoforms to develop molecules targeting tau aggregation is gaining significant attention in HTS efforts.One of these HTS studies using Tau1N4R revealed that certain polyphenolic moieties,such as myricetin,epicatechin 3-gallate or gossypetin exhibited anti-aggregational activity.This HTS also suggested the inhibitory potential for phenothiazines (azures A and B,or methylene blue) and porphyrins (hematin or phthalocyanines)but differing in the mode of action.Porphyrins targeted equally monomeric,oligomeric,and filamentous tau,and disrupted PFFs (Gerson et al.,2014).However,phenothiazines,such as methylene blue and leuco-methylthioninium bis(hydromethanesulfonate) (LMTM) exhibited the highest potential.Both molecules reduced tau aggregation by preventing protein-protein interactionsin vitroand in cellular models,and disrupted PFFs.To date,methylene blue and LMTM are some of the few tau anti-aggregational small molecules that have entered the Clinical Phase.In a Phase III study,LMTM monotherapy administration showed promising results in mild AD patients,moderately enhancing brain atrophy(Wilcock et al.,2018).Further clinical trials are still ongoing.Furthermore,recent HTS studies with Tau2N4R and Tau0N3R indicated that cyaninebased structures,as N744,exerted concentrationand post-translational modifications-dependent inhibitory and disrupting activities.Remarkably,multivalent variants of N744 showed increased inhibitory potential (Longhena et al.,2018;Wang et al.,2021).

Other polyphenolic structures,previously described as generic amyloid inhibitors,like epigallocatechin-3-gallate,baicalein,cannabidiol,or curcumin,were also repurposed to target tau aggregation and PFFs.Epigallocatechin-3-gallate appeared to be effective in targeting multiple binding sites,diverting tau aggregation to offpathway and non-toxic structures (Dominguez-Meijide et al.,2020).Baicalein also precluded aggregation driving the reaction to the formation of off-pathway non-toxic oligomers;in contrast,curcumin inhibited oligomer formation (Longhena et al.,2018;Dominguez-Meijide et al.,2020).Interestingly,curcumin bound more strongly Tau0N4R than Tau0N3R,suggesting that the R2 domain,which contains a microtubulebinding domain region,may play a key role in the inhibitory mechanism.Regarding cannabidiol,it delayed and inhibited aggregation by interacting mainly with tyrosine residues in the hexapeptide region,forming stable complexes.Additionally,non-phenolic generic anti-aggregational scaffolds have been repurposed to prevent tau aggregation.Anle138b,a pyrazole-derivative previously described to prevent α-synuclein aggregation,was effective in averting tau aggregation by binding to the aggregates and preventing the formation of toxic oligomers.When administered in mice,the compound decreased protein deposits and reduced neuronal loss (Dominguez-Meijide et al.,2020).Another rationally designed modulator of α-synuclein aggregation,CLR01,precluded tau polymerizationin vitroand cells,showing dose dependence and no heparin competition.In animal models,CLR01 reduced tau pathogenic forms,improving muscle strength and behavior(Longhena et al.,2018).Other types of natural molecules,such as isoquinolones (ANTC-15) or terpene-derivates(salannin or crocin),significantly impacted tau aggregation,reducing β-sheet content and aggregate-mediated toxicity.Interestingly,ANTC-15 prevented heparin-but not arachidonic acid-induced aggregation,suggesting different aggregation mechanisms or structures in the presence of these cofactors.Flavonoids(liquiritigenin or calycosin),have also been described as potent modulators of tau natural isoforms deposition,redirecting the reaction towards amorphous,non-toxic species,but lacking disaggregational capacity (Jouanne et al.,2017;Dominguez-Meijide et al.,2020).

All in all,tau anti-aggregational compounds share structural features with previously known modulators of amyloid formation: i) highly hydrophobic regions (either aromatic rings or large hydrocarbonated chains) with nitrogen and sulfur atoms,which are supposed to contribute to protein-binding,and ii) polar ramifications of the hydrophobic moiety,such as aldehyde and alcohol groups,that might interfere with hydrogen-bond networks that stabilize and facilitate amyloid formation.Nevertheless,the effectiveness of these molecules in preventing tau aggregation requires careful evaluation,including the study of tau-specificity to avoid generic interactors that might alter unwanted biological pathways.The inhibitory potential of a majority of molecules has been assessed by employing K18/K19 variants.These non-natural tau variants do not fully mimic the aggregation process of natural isoforms.Consequently,compounds that potently inhibit K18/K19 aggregation may not necessarily exhibit similar activity against natural isoforms due to differences in the pathway and,thus,in intermediate and fibrillar structures.Additionally,tau aggregation is typically induced in the presence of anionic cofactors like heparin.Although necessary to attain fast kinetics compatible with HTS strategies,these cofactors could also lead to false-positive results,either because the candidate interacts with the cofactor and not with the protein or because it induces an alternative aggregation pathway that does not recapitulate thein vivoprocess or conformers.Cryo-electron microscopy studies have revealed significant structural differences between tau patients andin vitrocofactor-induced fibrils (Shi et al.,2021).Aggregating tau in the absence of cofactors can partially mitigate these differences (Chakraborty et al.,2021),but as a general rule,one cannot assume that molecules with the capacity to target tauin vitrowould replicate their effectsin vivo.Cryo-electron microscopy structures of patients’fibrils can be used for the rational design of inhibitors,although they would be likely more effective in preventing fibril propagation than fibril formation.In any case,validating the inhibitory potential under more physiological conditions is an essential step in the development of potent and effective modulators of tau aggregation.Still,the use of truncated variants and cofactors could be beneficial in fast multiple-step discovery pipelines,which may involve: i) analysis of anti-aggregational properties of large chemical libraries in effectivetime frames using K18/K19,ii) validation of thein vitroactivity against natural isoforms,and iii) study of the activity in the absence of cofactorsin vitroor using cellular and animal models.

The potential of tau aggregation inhibitors to develop disease-modifying therapies for AD and other tauopathies is evident,but they are receiving less attention than those targeting other amyloidogenic proteins such as Aβ or α-synuclein.The main reason for this disparity is the complexity of the system.Investing resources to overcome these restraints can result in substantial progress in the battle against these debilitating diseases.

This work was funded by European Union Horizon 2020 research and innovation programme under GA 952334 (PhasAGE),by the Spanish Ministry of Science and Innovation (PID2019-105017RB-I00),and by ICREA,ICREA Academia 2015,and 2020 (to SV).

Zoe Manglano-Artuñedo,Samuel Peña-Díaz*,Salvador Ventura*

Institut de Biotecnologia i Biomedicina,Universitat Autònoma de Barcelona,Bellaterra,Spain

*Correspondence to:Samuel Peña-Díaz,PhD,Samuel.pdiaz@uab.cat;Salvador Ventura,PhD,Salvador.ventura@uab.cat.

https://orcid.org/0000-0002-2902-823X(Samuel Peña-Díaz)

Date of submission:April 28,2023

Date of decision:June 7,2023

Date of acceptance:June 17,2023

Date of web publication:July 20,2023

https://doi.org/10.4103/1673-5374.380900

How to cite this article:Manglano-Artuñedo Z,Peña-Díaz S,Ventura S (2024) Small molecules to target tau amyloid aggregation.Neural Regen Res 19(3):509-511.

Open access statement:This is an open access journal,and articles are distributed under the terms of the Creative Commons Attribution Non Commercial-Share Alike 4.0 License,which allows others to remix,tweak,and build upon the work non-commercially,as long as appropriate credit is given and the new creations are licensed under the identical terms.

Open peer reviewer:Joseph B.Rayman,Columbia University Irving Medical Center,USA.

Additional files:

Additional file 1:Open peer review report 1.

Additional file 2:Authors’ responses.