Perspective NEURAL REGENERATION RESEARCH www.nrronline.org Small molecules to target tau underlie the inhibitory activity, opening an avenue for developing nitrocatechol derivatives as tau amyloid aggregation 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. Zoe Manglano-Artuñedo, Samuel Peña-Díaz*, Salvador Ventura* Tau aggregation is driven by the two hexapeptide Protein aggregation has been linked with many strategies have been investigated: i) regulating motifs located at R2 and R3 repeats. However, Downloaded from http://journals.lww.com/nrronline by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCyw neurodegenerative diseases, such as Alzheimer’s tau hyperphosphorylation, ii) downregulating e v i d e n c e s u g g e st s t h at t wo n e i g h b o r i n g disease (AD) or Parkinson’s disease. AD belongs ta u ex p re s s i o n , i i i ) p ro m o t i n g c l e a ra n c e cysteines (C291 and C322) may play an important to a group of heterogeneous and incurable of phosphorylated tau, and iv) developing role in promoting aggregation by stablishing CX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 10/26/2023 neurodegenerative disorders collectively known modulators of tau amyloid aggregation (Zhang intermolecular disulphide bonds. This discovery as tauopathies. They comprise frontotemporal et al., 2022), including antibodies, peptides or prompted a rationale for the development dementia, Pick’s disease, or corticobasal small molecules. In this scenario, small molecules of novel inhibitors. A thiol-reactive rosamine degeneration, among others. The symptomatology are often preferred due to their enhanced derivative molecule, TR-2, has been reported to varies with the specific tau protein variant ability to penetrate the blood-brain barrier exhibit potent activity against K18 fibrillization by involved and the affected brain region or cell type. compared to protein-based interventions. For binding to and blocking cysteine residues, thus However, they share a common neuropathological these compounds, a detailed characterization of hindering the pathological tau aggregation (Gerson hallmark - the formation of proteinaceous deposits their inhibitory mechanism, the targeted species et al., 2014). The C291/C322 reactivity was named neurofibrillary tangles. Neurofibrillary (oligomers, protofibrils, and/or fibrils), and a also employed to develop a strategy to identify tangles, primarily composed of aggregated tau validation of their in vivo effect are imperative covalent inhibitors of K18 fibrillization. As a result, (Zhang et al., 2022), disrupt normal neuronal before they can be evolved into effective drugs for vinylsulfone-derivates were found to interfere with functions, leading to cell death and cognitive clinical use. the aggregation process. These findings suggest decline. that the covalent binding of molecules to cysteine The disordered nature of tau has posed challenges residues near the hexapeptide-amyloid motifs can Tau is an intrinsically disordered protein that for drug development using structure-based modulate tau aggregation (Petri et al., 2022). contributes to microtubule stabilization and approaches. Methodologies such as high- plays an important role in axonal transport. The The combination of drug repositioning and throughput screening (HTS) or drug repurposing, tau protein sequence can be dissected into four rational design resulted in the repurposing of two have emerged as alternatives to identify molecules domains: i) the amino-terminal domain that hybrid molecules, naphtoquinone-tryptophan with inhibitory properties against tau aggregation. contains up to two amino acid inserts (N1 and N2); and naphtoquinone-dopamine as tau aggregation Over the years, both strategies have rendered ii) a proline-rich segment; iii) the microtubule- inhibitors. Both molecules were designed based a variety of modulators targeting aberrant tau binding domain, a positively-charged region on the critical role of aromatic residues in polymerization (Figure 1B). that encloses 3/4 imperfect repeats (R1, R2, R3, stabilizing amyloid fibril structures. The quinone and R4), and delineates two aggregation-prone In recent years, HTS protocols using amyloid skeleton was selected for its reported potential hexapeptides (at R2 and R3); and iv) the carboxy- dyes like thioflavin-T or -S have been exploited to to inhibit the self-assembly of different amyloid terminal domain. Remarkably, alternative splicing discover fibrillization inhibitors of tau truncated proteins, and tryptophan/dopamine molecules gives rise to six different isoforms that vary variants, which exhibit accelerated aggregation were expected to bind the aromatic residues, depending on the presence or absence of R2 (K18/ kinetics. In this way, the screening of ~51,000 interfering with the aromatic π-π interactions K19 fragment, respectively) and of the N inserts compounds against K18 aggregation led to the that sustain the amyloid core. Naphtoquinone- (Figure 1A). Additionally, tau also undergoes discovery of 113F08 and 330B06, which contain tryptophan inhibited the aggregation of one of numerous post-translational modifications, a 2,3-di(furan2-yl)-quinoxaline structure, as the tau hexapeptides and ameliorated tauopathy- i n c l u d i n g a c e t y l at i o n , u b i q u i t i n at i o n , o r concentration-dependent modulators of K18 related phenotype in an animal model expressing phosphorylation, which has been proposed to aggregation. In another strategy, the development human tau. In contrast, naphtoquinone-dopamine play a key role in tau function and aggregation. of a ligand-based computational screening, inhibited the aggregation and disassembled In pathological conditions, hyperphosphorylation based on pharmacophore models, identified the PFFs of both hexapeptide motifs and Tau2N4R reduces tau-microtubule affinity and promotes phenylthiazol-hydrazide scaffold as a source of (Dominguez-Meijide et al., 2020). Interestingly, aggregation. Tau aggregation is a multifaceted inhibitors of K19 aggregation. Another HTS study these molecules have been found to efficiently process that involves the generation of various analyzed the activity of different molecules against cross the blood-brain barrier, offering a potential species. The self-assembly of phosphorylated tau K19 heparin-induced aggregation. Compounds therapeutic application in AD. monomers initiates the formation of oligomeric like B4D5 or B4D3 significantly decreased the K18/K19 variants have been used to pre-screen species that are often enriched in β-sheet formation of amyloids and even disrupted pre- the inhibitory activity of anti-aggregational architecture and expose hydrophobic patches to formed fibrils (PFFs). Importantly, cell models compounds, subsequently assayed on top of tau the solvent. These arrangements are thought to based on the K18 variant suggested a protective natural variants. Certain modulators displayed be the most cytotoxic structures in amyloidosis. activity against aggregate-mediated toxicity a polyphenol-scaffold, including PE859, which Over time, the oligomeric species evolve into (Longhena et al., 2018). prevented K18/K19 and 2N4R aggregation protofibrillar assemblies, which ultimately mature Drug repositioning has emerged as an alternative and showed efficacy in mouse models, and into highly structured fibrils. Noticeably, these to HTS, as it reduces the risk of failure, the oleocanthal, which prevented K18 and Tau2N4R fibrils can induce the deposition of soluble tau in time necessary to reach the market, and the aggregation by covalently binding to lysine residues healthy neurons, leading to the propagation of cost. For instance, tolcapone and entacapone, on the hexapeptide repeat via its aldehyde groups pathological amyloids throughout the brain (Zhang two catechol-O-methyltransferase inhibitors (Dhouafli et al., 2018; Longhena et al., 2018; et al., 2022). prescribed for Parkinson’s disease treatment, Wang et al., 2021). Similarly, anthraquinones such In the quest to halt or slow down the progression exhibited anti-aggregating properties against tau as emodin or asperbenzaldehyde, which share of tauopathies, inhibiting tau aggregation has hexapeptides fibrillization. Notably, both drugs a tricyclic aromatic ring scaffold, were identified emerged as a promising approach. Multiple share a nitrocatechol scaffold that is believed to as tau aggregation inhibitors in an HTS using K19 NEURAL REGENERATION RESEARCH｜Vol 19｜No. 3｜March 2024｜509 NEURAL REGENERATION RESEARCH www.nrronline.org Perspective also suggested the inhibitory potential for A 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 Downloaded from http://journals.lww.com/nrronline by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCyw the highest potential. Both molecules reduced tau aggregation by preventing protein-protein interactions in vitro and in cellular models, and CX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 10/26/2023 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 B ongoing. Furthermore, recent HTS studies with Tau2N4R and Tau0N3R indicated that cyanine- based structures, as N744, exerted concentration- and 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 off- pathway 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 microtubule- binding domain region, may play a key role in the Figure 1 ｜ Overview of tau sequence and anti-aggregation moieties. inhibitory mechanism. Regarding cannabidiol, it (A) Schematic representation of the domains of the different tau isoforms, including natural variants (2N4R, 1N4R, 0N4R, 2M3R, 1N3R, 0N3R) and artificial constructs (K18, K19). (B) Structure of some tau anti-aggregational compounds. delayed and inhibited aggregation by interacting In yellow, compounds whose inhibitory potential has been analyzed with artificial constructs. In green, chemicals that mainly with tyrosine residues in the hexapeptide were tested to inhibit both, artificial and natural variants of the protein. In blue, molecules whose anti-aggregational activity was analyzed using only natural variants. Created with BioRender.com and Microsoft® PowerPoint® for Microsoft region, forming stable complexes. Additionally, 365 MSO, version 2304. EGCG: Epigallocatechin-3-gallate; LMTM: leuco-methylthioninium bis(hydromethanesulfonate); non-phenolic generic anti-aggregational scaffolds NQDA: naphtoquinone-dopamine. have been repurposed to prevent tau aggregation. Anle138b, a pyrazole-derivative previously and further validated in 0N3R and 0N4R variants. molecules were the worst inhibitors of natural- described to prevent α-synuclein aggregation, was Interestingly, anthraquinones also inhibited occurring variants aggregation compared to K18/ effective in averting tau aggregation by binding K18∆K280 aggregation, which does not require K19, suggesting potential variations in the mode to the aggregates and preventing the formation heparin for fibrillization, demonstrating that the of action for truncated and naturally occurring of toxic oligomers. When administered in mice, inhibitory activity did not rely on compound- isoforms. the compound decreased protein deposits and heparin interactions (Gerson et al., 2014; reduced neuronal loss (Dominguez-Meijide et al., Longhena et al., 2018). Another HTS exploiting Not surprisingly, the exclusive use of natural 2020). Another rationally designed modulator of K18P301L aggregation led to the discovery of isoforms to develop molecules targeting tau α-synuclein aggregation, CLR01, precluded tau aminothienopyridazynes, which exhibited anti- aggregation is gaining significant attention in HTS polymerization in vitro and cells, showing dose aggregating features without interfering with efforts. One of these HTS studies using Tau1N4R dependence and no heparin competition. In tau-mediated microtubules assembly and an revealed that certain polyphenolic moieties, such animal models, CLR01 reduced tau pathogenic inhibitory effect in 2N4R (Gerson et al., 2014). as myricetin, epicatechin 3-gallate or gossypetin forms, improving muscle strength and behavior Remarkably, despite effectiveness, most of these exhibited anti-aggregational activity. This HTS (Longhena et al., 2018). 510 ｜NEURAL REGENERATION RESEARCH｜Vol 19｜No. 3｜March 2024 Perspective NEURAL REGENERATION RESEARCH www.nrronline.org Other types of natural molecules, such as effective in preventing fibril propagation than fibril References isoquinolones (ANTC-15) or terpene-derivates formation. In any case, validating the inhibitory Chakraborty P, Riviere G, Liu S, de Opakua AI, Dervisoglu R, (salannin or crocin), significantly impacted tau potential under more physiological conditions is an Hebestreit A, Andreas LB, Vorberg IM, Zweckstetter M aggregation, reducing β-sheet content and essential step in the development of potent and (2021) Co-factor-free aggregation of tau into seeding- aggregate-mediated toxicity. Interestingly, ANTC- effective modulators of tau aggregation. Still, the competent RNA-sequestering amyloid fibrils. Nat 15 prevented heparin- but not arachidonic use of truncated variants and cofactors could be Commun 12:4231. acid-induced aggregation, suggesting different beneficial in fast multiple-step discovery pipelines, Dhouafli Z, Cuanalo-Contreras K, Hayouni EA, Mays ag gregation mechanisms or structures in which may involve: i) analysis of anti-aggregational CE, Soto C, Moreno-Gonzalez I (2018) Inhibition of the presence of these cofactors. Flavonoids properties of large chemical libraries in effective Downloaded from http://journals.lww.com/nrronline by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCyw protein misfolding and aggregation by natural phenolic (liquiritigenin or calycosin), have also been time frames using K18/K19, ii) validation of the in compounds. Cell Mol Life Sci 75:3521-3538. described as potent modulators of tau natural vitro activity against natural isoforms, and iii) study Dominguez-Meijide A, Vasili E, Outeiro TF (2020) isoforms deposition, redirecting the reaction of the activity in the absence of cofactors in vitro CX1AWnYQp/IlQrHD3i3D0OdRyi7TvSFl4Cf3VC1y0abggQZXdgGj2MwlZLeI= on 10/26/2023 Pharmacological modulators of tau aggregation and towards amorphous, non-toxic species, but lacking or using cellular and animal models. spreading. Brain Sci 10:858. disaggregational capacity (Jouanne et al., 2017; Gerson JE, Castillo-Carranza DL, Kayed R (2014) Advances The potential of tau aggregation inhibitors to Dominguez-Meijide et al., 2020). in therapeutics for neurodegenerative tauopathies: develop disease-modifying therapies for AD All in all, tau anti-aggregational compounds and other tauopathies is evident, but they are moving toward the specific targeting of the most toxic share structural features with previously known receiving less attention than those targeting other tau species. ACS Chem Neurosci 5:752-769. modulators of amyloid formation: i) highly amyloidogenic proteins such as Aβ or α-synuclein. Jouanne M, Rault S, Voisin-Chiret AS (2017) Tau protein hydrophobic regions (either aromatic rings or The main reason for this disparity is the complexity aggregation in Alzheimer’s disease: an attractive target large hydrocarbonated chains) with nitrogen and of the system. Investing resources to overcome for the development of novel therapeutic agents. Eur J sulfur atoms, which are supposed to contribute to these restraints can result in substantial progress Med Chem 139:153-167. protein-binding, and ii) polar ramifications of the in the battle against these debilitating diseases. Longhena F, Spano P, Bellucci A (2018) Targeting hydrophobic moiety, such as aldehyde and alcohol of disordered proteins by small molecules in groups, that might interfere with hydrogen-bond This work was funded by European Union Horizon neurodegenerative diseases. Handb Exp Pharmacol networks that stabilize and facilitate amyloid 2020 research and innovation programme under 245:85-110. formation. Nevertheless, the effectiveness of GA 952334 (PhasAGE), by the Spanish Ministry of Mohamed T, Hoang T, Jelokhani-Niaraki M, Rao PP (2013) these molecules in preventing tau aggregation Science and Innovation (PID2019-105017RB-I00), Tau-derived-hexapeptide 306VQIVYK311 aggregation requires careful evaluation, including the study and by ICREA, ICREA Academia 2015, and 2020 (to inhibitors: nitrocatechol moiety as a pharmacophore in of tau-specificity to avoid generic interactors that SV). drug design. ACS Chem Neurosci 4:1559-1570. might alter unwanted biological pathways. The Petri L, Abranyi-Balogh P, Vagrys D, Imre T, Varro N, inhibitory potential of a majority of molecules Zoe Manglano-Artuñedo, Mandity I, Racz A, Wittner L, Toth K, Toth EZ, Juhasz T, has been assessed by employing K18/K19 Samuel Peña-Díaz*, Salvador Ventura* Davis B, Keseru GM (2022) A covalent strategy to target Institut de Biotecnologia i Biomedicina, Universitat intrinsically disordered proteins: discovery of novel tau variants. These non-natural tau variants do not Autònoma de Barcelona, Bellaterra, Spain fully mimic the aggregation process of natural aggregation inhibitors. Eur J Med Chem 231:114163. *Correspondence to: Samuel Peña-Díaz, PhD, isoforms. Consequently, compounds that potently Shi Y, Zhang W, Yang Y, Murzin AG, Falcon B, Kotecha A, Samuel.pdiaz@uab.cat; Salvador Ventura, PhD, inhibit K18/K19 aggregation may not necessarily Salvador.ventura@uab.cat. van Beers M, Tarutani A, Kametani F, Garringer HJ, exhibit similar activity against natural isoforms https://orcid.org/0000-0002-2902-823X Vidal R, Hallinan GI, Lashley T, Saito Y, Murayama S, due to differences in the pathway and, thus, in (Samuel Peña-Díaz) Yoshida M, Tanaka H, Kakita A, Ikeuchi T, Robinson intermediate and fibrillar structures. Additionally, Date of submission: April 28, 2023 AC, et al. (2021) Structure-based classification of tau aggregation is typically induced in the presence Date of decision: June 7, 2023 tauopathies. Nature 598:359-363. of anionic cofactors like heparin. Although Date of acceptance: June 17, 2023 Wang L, Bharti, Kumar R, Pavlov PF, Winblad B (2021) Date of web publication: July 20, 2023 Small molecule therapeutics for tauopathy in necessary to attain fast kinetics compatible with HTS strategies, these cofactors could also Alzheimer’s disease: walking on the path of most https://doi.org/10.4103/1673-5374.380900 lead to false-positive results, either because the resistance. Eur J Med Chem 209:112915. How to cite this article: Manglano-Artuñedo Z, candidate interacts with the cofactor and not with Wilcock GK, Gauthier S, Frisoni GB, Jia J, Hardlund JH, Peña-Díaz S, Ventura S (2024) Small molecules to the protein or because it induces an alternative target tau amyloid aggregation. Neural Regen Res Moebius HJ, Bentham P, Kook KA, Schelter BO, Wischik aggregation pathway that does not recapitulate 19(3):509-511. DJ, Davis CS, Staff RT, Vuksanovic V, Ahearn T, Bracoud L, the in vivo process or conformers. Cryo-electron Open access statement: This is an open Shamsi K, Marek K, Seibyl J, Riedel G, Storey JMD, et al. microscopy studies have revealed significant access journal, and articles are distributed (2018) Potential of low dose leuco-methylthioninium structural differences between tau patients and under the terms of the Creative Commons bis(hydromethanesulphonate) (LMTM) monotherapy in vitro cofactor-induced fibrils (Shi et al., 2021). AttributionNonCommercial-ShareAlike 4.0 License, for treatment of mild Alzheimer’s disease: cohort which allows others to remix, tweak, and build analysis as modified primary outcome in a phase III Aggregating tau in the absence of cofactors can upon the work non-commercially, as long as clinical trial. J Alzheimers Dis 61:435-457. partially mitigate these differences (Chakraborty appropriate credit is given and the new creations et al., 2021), but as a general rule, one cannot Zhang Y, Wu KM, Yang L, Dong Q, Yu JT (2022) are licensed under the identical terms. assume that molecules with the capacity to target Tauopathies: new perspectives and challenges. Mol Open peer reviewer: Joseph B. Rayman, Columbia tau in vitro would replicate their effects in vivo. University Irving Medical Center, USA. Neurodegener 17:28. Cryo-electron microscopy structures of patients’ Additional files: fibrils can be used for the rational design of Additional file 1: Open peer review report 1. P-Reviewer: Rayman JB; C-Editors: Zhao M, Liu WJ, Qiu Y; inhibitors, although they would be likely more Additional file 2: Authors’ responses. T-Editor: Jia Y NEURAL REGENERATION RESEARCH｜Vol 19｜No. 3｜March 2024｜511