Tau protein and tau aggregation inhibitors

Tau protein and tau aggregation inhibitors

Neuropharmacology 59 (2010) 276e289 Contents lists available at ScienceDirect Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm...

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Neuropharmacology 59 (2010) 276e289

Contents lists available at ScienceDirect

Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm


Tau protein and tau aggregation inhibitors Bruno Bulic a,1, Marcus Pickhardt b, Eva-Maria Mandelkow b, Eckhard Mandelkow b, * a b

Center for Advanced European Studies and Research, Ludwig-Erhard-Allee 2, 53175 Bonn, Germany Max-Planck-Unit for Structural Molecular Biology, 22607 Hamburg, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 October 2009 Received in revised form 20 January 2010 Accepted 26 January 2010

Alzheimer disease is characterized by pathological aggregation of two proteins, tau and Ab-amyloid, both of which are considered to be toxic to neurons. In this review we summarize recent advances on small molecule inhibitors of protein aggregation with emphasis on tau, with activities mediated by the direct interference of self-assembly. The inhibitors can be clustered in several compound classes according to their chemical structure, with subsequent description of the structureeactivity relationships, showing that hydrophobic interactions are prevailing. The description is extended to the pharmacological profile of the compounds in order to evaluate their drug-likeness, with special attention to toxicity and bioavailability. The collected data indicate that following the improvements of the in vitro inhibitory potencies, the consideration of the in vivo pharmacokinetics is an absolute prerequisite for the development of compounds suitable for a transfer from bench to bedside. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Aggregation inhibitors Alzheimer's disease Amyloids Neurodegeneration Tau protein

1. Introduction In protein aggregation diseases such as Alzheimer, Parkinson, Huntington and others, it is known that aggregation inhibitors of a specific amyloidogenic peptide are also potential aggregation inhibitors of a wide variety of other amyloidogenic peptides (Heiser et al., 2002; Jin et al., 2003; Porat et al., 2006; Taniguchi et al., 2005). Histological dyes such as Thioflavin S or Congo red bind to aggregates in at least fifteen genetically unrelated disorders (Kelly, 1996). Indeed, amyloid fibrils, irrespective of their amino acid sequence, share a common X-ray diffraction pattern showing a characteristic 4.6e4.8 Å meridional reflection (Fig. 1). It corresponds to the spacing between the peptide chains forming cross-b structure, such that the b-strands of the proteins are arranged perpendicular to the fibril axis (Fig. 1b). AFM microscopy of Ab42 aggregates in reconstituted membranes revealed a remarkable supramolecular ionchannel like structure, (Lashuel and Lansbury, 2006; Quist et al., 2005), a striking feature shared by most amyloids such as a-synuclein, Ab42, IAPP and others. Irrespective of their amino acid sequence, some of the soluble oligomeric amyloids also have similar structures, which are recognised by oligomer-specific antibodies independently of the amino acid sequence (Glabe, 2004;

* Corresponding author. c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany. Fax: þ49 (0) 40 89716810. E-mail addresses: [email protected] (B. Bulic), [email protected] (E. Mandelkow). 1 Fax: þ49 22896569401. 0028-3908/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2010.01.016

Kayed et al., 2007, 2003). It is noteworthy that even when a compound class is reported to be inhibitor of a specific amyloid type, its inhibitory potency on other amyloid types can hardly be predicted, due to variations in the interactions with the amino acid residues forming the peptide backbone. Neurodegenerative diseases associated with tau proteins which self-assemble into abnormal fibers (“paired helical filaments” or PHFs) are known as “tauopathies” (Goedert et al., 1998). These filaments can form higher order aggregates (“neurofibrillary tangles”, “neuropil threads”) in neurons or other cell types of the brain (Ballatore et al., 2007; Kidd, 1963; Leroy et al., 2007; Sawaya et al., 2007; Vieira et al., 2007). PHFs have the appearance of two filaments twisted around one other with a cross-over repeat of 80 nm and an apparent width varying between w10 and w22 nm. The most common tauopathy is Alzheimer's disease, but tau deposits also occur in frontotemporal dementias (FTDP-17), Pick's disease, Parkinson's disease, progressive nuclear palsy and other conditions (Arima, 2006; Guerrero et al., 2008). The neurodegenerative processes involved in AD are still poorly understood, however, protein aggregates and oligomers are recognised as major elements of cellular toxicity. Therefore, small molecule inhibitors of protein aggregation are anticipated drug candidates, and preliminary data on a phase II clinical trial with the drug candidate methylene blue chloride (MTC) has been claimed to support the concept of tau aggregation inhibition as a means to address Alzheimer's disease (Wischik et al., 1996, 2007). A significant advance in understanding tau's behaviour came when it was recognised that the protein contains isolated short

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Fig. 1. a) X-ray diffraction pattern of PHFs derived from the tau construct K18 reprinted from von Bergen et al. ref (von Bergen et al., 2001); b) schematic representation of an amyloid protofibril (Serpell et al., 2007).

peptide motifs, embedded in an otherwise hydrophilic environment, which have a high tendency for b-structure and aggregation (von Bergen et al., 2000). Indeed, two such motifs lie within the “repeat domain” of tau (repeat 2 and repeat 3) which is known to be responsible for aggregation, forming the core of the paired helical filaments. As a direct test of this view, any mutation in the hexapeptide motifs that disrupts b-structure (e.g. an inserted proline) renders the protein incompetent for assembly. Conversely, mutations that enhance the b-propensity of the motifs also enhance the aggregation of tau; this includes at least two of the tau mutations found in FTDP-17 (von Bergen et al., 2001). Interestingly, we found that the same motifs are involved not only in abnormal tau aggregation but also in microtubule interactions, as seen by NMR spectroscopy (Mukrasch et al., 2005). This illustrates the close relationship between pathological and physiological functions of tau. These studies provide structural insights into the aggregation process and open avenues to inhibit it by suitable compounds. Such compounds could be developed into drugs for treating the neurofibrillary pathology of Alzheimer's disease and related forms of degeneration (Klunk et al., 2002). Note that tau is a natively unfolded protein lacking a defined 3D structure; it therefore does not lend itself to X-ray structure analysis, but the protein and several of its domains have been analyzed by NMR spectroscopy (Mukrasch et al., 2009, 2007a,b). The interplay between Ab aggregation and tau toxicity has been recently stressed in the literature (King et al., 2006; Park and Ferreira, 2005; Rapoport et al., 2002; Roberson et al., 2007; Small and Duff, 2008; Talaga and Quere, 2002). Therefore, addressing only Ab aggregates without considering tau aggregates might not yield the expected cognitive improvements. This was demonstrated by the follow-up studies on the discontinued immunisation therapy with AN-1792, which showed an effective clearance of the brain Ab-amyloid load without significant cognitive improvements (Ikonomovic et al., 2008). Two major approaches are distinguishable for addressing tau aggregation. The first is the search for inhibitors of kinases that

phosphorylate tau, based on the assumption that abnormally phosphorylated tau aggregates more readily (Kelleher et al., 2007; Kosik and Shimura, 2005). The second approach is the search for direct inhibitors of the tau aggregation process. Here we will focus on the second approach. One straightforward strategy for the discovery of new active molecules is the screening of compound libraries containing sufficient structural diversity. Identification of hits generally enables the subsequent medicinal chemistry efforts necessary to arrive at compounds displaying the desired properties, i.e. optimized inhibitory potencies and pharmacokinetics. Regarding methods, several approaches have been used to perform high or medium throughput screens for aggregation inhibitors. They include fluorescence-based assays of Tau aggregation using thioflavin S as an indicator (Pickhardt et al., 2005a), filter assays to segregate soluble and insoluble protein (Heiser et al., 2002; Chang and Kuret, 2008), sarkosyl treatment and quantification of sarkosylsoluble and insoluble tau protein (Taniguchi et al., 2005), quantification of filament length and number by transmission electron microscopy (Congdon et al., 2007), and fluorescence polarization of labelled Tau which distinguishes diffusible from aggregated molecules (Crowe et al., 2009). 2. Identified tau aggregation inhibitors To identify potential tau aggregation inhibitors we used the tau construct K19 which contains the three repeat domain of the foetal human isofom hTau23 for the high throughput assay (Fig. 2). The construct corresponds to the b-sheet forming core of the PHF structure including the VQIVYK hexapeptide motif that promotes aggregation (von Bergen et al., 2000). The screening was based on the thioflavin S fluorescence assay (Friedhoff et al., 1998) where the emission of thioflavin S is strongly increased at 521 nm (excitation: 440 nm) when the dye is bound to ordered b-sheet structures. Thus the signal is proportional to the extent of aggregation. 200.000 compounds selected according to Lipinski's rules (Lipinski et al., 2001) were screened both for inhibition of tau aggregation and


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Fig. 2. Diagram of the full-length tau isoform htau40 and the repeat domain construct used in the PHF inhibition assay (construct K19, repeat domain with 3 repeats, R2 absent). PHF6 is the hexapeptide motif that nucleates the aggregation of construct K19.

for induced disassembly of tau aggregates (Pickhardt et al., 2005a,b). Tau protein was incubated overnight under conditions favoring aggregation in the presence of the screened compound (Fig. 3, upper part). The amount of aggregated tau protein was then quantified by fluorescence after addition of thioflavine S. In a similar approach the disassembly of tau fibrils was performed by incubation of preformed PHFs in the presence or absence of compounds overnight at 37  C and then measuring the ThS-fluorescence of the remaining fibrils (Fig. 3, lower part). The obtained Z-factor value of 0.81 confirmed the reliability of the assay (Zhang et al., 1999). The results were verified with other assays (electron microscopy, filter/pelleting-assay) as well as with other tau constructs and isoforms. The property of phosphorylated tau to detach from microtubules and to aggregate into fibrils depends on a complex phosphorylation pattern. In fact, phosphorylation could either contribute or hinder the aggregation, depending on its extent and localisation. Therefore the screening was performed with unphosphorylated recombinant tau which provided fibers highly similar to those purified from Alzheimer brain tissues and whose structure has been previously assessed by electron microscopy and

Fig. 3. Thioflavin S (ThS) fluorescence assay for compounds that inhibit aggregation of tau into PHFs (upper part), and compounds that disaggregate preformed PHFs (lower part) (Bulic et al., 2007).

spectroscopic methods, indicating that the in vitro aggregation is a reliable PHF model (Barghorn et al., 2004; Schneider et al., 1999). To avoid false-positive ThS-signals (and therefore false inhibitory effects) the compounds were initially screened for their potential self-fluorescence at the given excitation- and emission wavelengths. All compounds which showed a higher fluorescence signal in absence of protein than in the presence were excluded from further testing. To exclude possible quenching or ThSdisplacement effects the substances were also tested in 'dye-free' assays such as pelleting and filter assays, intrinsic tryptophan fluorescence or electron microscopy. The ability not only to inhibit tau aggregation but also to disassemble preformed aggregates was included in the selection parameters for the compound screen, leading to the elimination of 99.96% of the library compounds. The identified hits were able to induce the disassembly of PHFs with up to 80% efficiency at 60 mM compound concentration. These hits were classified in clusters according to their chemical structures. Details on the inhibitory activities obtained from the N-phenylamines, anthraquinones, phenylthiazolyl-hydrazides (PTHs) and thioxothiazolidinones (rhodanines) were described previously (Bulic et al., 2007; Khlistunova et al., 2007; Larbig et al., 2007; Pickhardt et al., 2007a, 2005b). A chemistry primarily aimed at elucidating the requirements for improved inhibitory potencies allowed the deduction of the structureeactivity relationship (SAR) of the two last mentioned compound clusters (PTHs and rhodanines). Selected compounds were subsequently tested on a neuronal cell model of tau aggregation (Fig. 4). 2.1. Rhodanine-based inhibitors The rhodanine based compounds (Fig. 5) are members of an appealing hit class. The scaffold is found in various bioactive compounds that were reported as antimalarial, antituberculous, antibiotic, anticancer, antitoxin and hypoglycaemic by targeting respectively the Enoyl-acyl Carrier Protein Reductase, peptide deformylase (PDF), RNA-polymerase, JNK-stimulating phosphatase 1 (JSP-1), bacterial proteases, PPAR-receptor and GSK-3 (Ahn et al., 2006; Cutshall et al., 2005; Gualtieri et al., 2006; Hu et al., 2008; Irvine et al., 2008; Johnson et al., 2008; Kumar et al., 2007; Martinez et al., 2005; Russell et al., 2009; Villain-Guillot et al., 2007; Zervosen et al., 2004). When focusing on Alzheimer's disease, the pleiotropic effect of rhodanines might be of interest since both PPARg and GSK-3 are potential targets in AD. Although derivatives are suspected to undergo conjugate addition in vivo that might reduce their plasma half-life, (Carlson et al., 2006) they are frequently employed in medicinal chemistry with no apparent side effects. Their bioavailability and tolerability are reported in a long-term clinical study with epalrestat (3 years, 150 mg/day oral administration), an aldose reductase inhibitor indicated for diabetic neuropathy (Fig. 6) (Hotta et al., 2006). The in vitro permeability (rat jujenum) was observed at Papp ¼ 4.34  1.78  10e6 cm/s. The extracted data indicate a good permeability with an excellent bioavailability (100  22% bioavailability, peak plasma concentration of 4.0  0.9 mg/ml at 1.7 h after administration of a 50 mg oral dose to adults) and a good plasma stability (half-life t1/2: 102.5 min) (Sturm et al., 2006). Epalrestat was found to be highly protein bound in plasma with a 90% binding rate. Derivatives based on the rhodanine scaffold were submitted to in vitro pharmacodynamic analyses, showing their adequate permeability, non-toxicity on HepG2 cells, moderate effect on cytochrome P450s and hERG, but variable stability with liver microsomes, depending on the rhodanine substituents (Johnson et al., 2008). On the other side, the parent thiazolidinedione heterocycle present in PPAR agonists such has troglitazone is associated with high

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Fig. 4. Tau expression, aggregation and inhibition in a cellular model affected by compounds (Larbig et al., 2007; Pickhardt et al., 2007b; Bulic et al., 2007).

hepatotoxicity that might be however related to the substitutents since pioglitazone and rosiglitazone are considered safe (Fig. 6) (Park et al., 2005; Tolman and Chandramouli, 2003). The rhodanine core for tau aggregation inhibition has been investigated via the synthesis of a focused library. In these experiments, rhodanines (R1 ¼ S and R2 ¼ S, Fig. 6b), thiohydantoin (R1 ¼ S and R2 ¼ N), thioxooxazolidine (R1 ¼ S and R2 ¼ O), oxazolidinedione (R1 ¼ O and R2 ¼ O), and hydantoin (R1 ¼ O and R2 ¼ N) were synthesised and screened for activity on tau aggregation inhibition and disaggregation of preformed tau aggregates. The following trend in the depolymerisation of tau aggregates was observed: rhodanine (IC50/DC50 (mM); 0.8/0.1) > thiohydantoin (6.1/0.4) >> oxazolidinedione (3.5/2.2) ¼ thioxooxazolidinone (3.1/ 2.4) >> hydantoin (22.6/54.3). The IC50 and DC50 values represent respectively the assembly-inhibiting and disassembly-inducing half-maximal concentrations measured in vitro. The rhodanine heterocycle appeared to be the most potent, underlining the importance of the thioxo group in rhodanines. The rhodanine core has been reported as a carboxylic acid bioisoster by size, low electronegativity, and ability to engage in hydrogen bonds (Patani and LaVoie, 1996).

Fig. 5. Variation of rhodanine inhibitor structure. Illustration of the variations on the core (R1 and R2) and on the flanking substituents (R3 and R4) (Bulic et al., 2007).

Besides the central rhodanine core, the substitution patterns on R1 and R3 (Fig. 5) showed that hydrogen bond acceptors in the form of a nitro group, carboxylic acids, phenols, sulfonates/sulfonamides are required, in line with observations from other known amyloid aggregation inhibitors (Bulic et al., 2007). Noteworthy, the total length of the molecule proved to be of importance, as also reported below (par. 2.3) for curcumin. Variations of the length of the linker between the carboxylic acid and the rhodanine core (red part, Fig. 5) revealed that increasing the distance up to two carbon bonds resulted in an appreciable increase in the compound's inhibitory potency indicating an optimal positioning of the inhibitor toward its binding site. The heteroaromatic side chain (R3, Fig. 5) tolerated variations, but modifications on the furan heterocycle showed that the electron density distribution on the molecule is important, as replacement of the furan ring in 16 for thiophene in 22 (Fig. 7) lowered the potency, as well as the pyridine in 27 compared to the furan in 19. Moreover, the presence of an aromatic side chain was important, supporting an hydrophobic interaction and/or p-stacking on this fragment (Waters, 2002). After optimization, an interesting 170 nM IC50 (compound 19) could be obtained. The compounds were further tested on neuronal cell models of tau aggregation, with activities depicted in Fig. 12. This also illustrated the slight discrepancy between potencies observed in vitro and in the cell-based assay, reflecting the need for further optimization of this compound class with respect to ADME parameters (absorption/distribution/metabolism/excretion) relevant for the in vivo activity, and in the first place their membrane permeability for cell models. Whereas the toxicity was restricted to a safe range (2e8%, LDH assay in N2A cells, incubation 24 h with 10 mM compound, Fig. 12), the negatively charged carboxylate present on most compounds may limit the membrane permeability. The better efficiency on cells of charge-neutral benzimidazole-containing


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Fig. 6. Rhodanine inhibitor of tau aggregation (01) and parent drug compounds.

compound 14 (Fig. 7) might therefore be correlated with its better permeability, a feature that would allow optimization of the other inhibitors for in vivo potency by modification of the carboxylate with charge-neutral bioisosters. Because a discussion about toxicity of small oligomers has emerged there may be concerns about the toxicity of fragments generated by the disassembly of fibrils (Lashuel and Grillo-Bosch, 2005; Necula et al., 2007). Nevertheless, studies using compound 14 allowed observing the effect of tau filament disassembly on cellular viability. A clear reversal of the toxicity caused by tau aggregation in the cytosol was seen (Khlistunova et al., 2007). Also, recent reports on compounds binding to Ab fibrils indicated that they might also bind to oligomers and further support neuroprotective effects (Hong et al., 2007; Maezawa et al., 2008). 2.2. Phenylthiazolyl-hydrazide inhibitors In parallel to the rhodanine compounds, a hit from HTS-screen was selected for lead optimization and SAR investigation (Larbig et al., 2007). The application of in silico scaffold hopping provided the phenylthiazolyl-hydrazide (PTH) scaffold that was further developed through synthetic derivatisation of R1eR3 (Fig. 8) in order to improve its aggregation inhibition potency. This is in agreement with the pharmacophore model obtained by scaffold hopping which stipulated two aromatic rings at R1 and R3, a hydrophobic region on the thiazole ring and a hydrogen bond acceptor on the carboxyl amide (Fig. 8). Through the implementation of a hydrogen binding substituent on R3 a further improvement of the inhibitory potency were observed, e.g. in BSc3094 (Fig. 9). In parallel, the hydrophobic or pstacking interactions on R1 appeared to contribute to target binding, as confirmed by STD-NMR experiments revealing a strong interaction of the tau construct K18 with the R1 aromatic ring (Fig. 9). Moreover, the STD-NMR experiments showed that the binding is specific with 62 mM dissociation constant (Pickhardt et al., 2007b). The comparison of in vitro activities of compounds must consider possible differences in cell permeabilities which can balance their efficiency in cells, e.g. the PTHs compared to the above-mentioned rhodanines. Their activity in cells appeared to be in the same range than rhodanines as depicted in Fig. 12. Nevertheless, these phenylthiazolyl-hydrazides also had a somewhat higher cytotoxicity (Fig. 12), in good agreement with hydrazidecontaining substances from the literature. Indeed, numerous substances presenting the thiazolyl-hydrazides scaffold are reported with analgesic, antibacterial, antifungal and antiproliferative activities (Hafez and El-Gazzar, 2008; Sonar and Crooks, 2009; Vijaya Raj et al., 2007). The mode of actions and targets are not entirely identified, although their antimalarial activity is thought to be due to the inhibition of the cysteine protease cruzain (Leite et al., 2006). Focusing on the thiazole moiety (in blue in Fig. 10), a Monoamine Oxidase (MAO) irreversible inhibition has been reported (Raciti et al., 1995). Thiazoles are widespread heterocycles,

commonly observed in medicinal chemistry and natural compounds. The most prominent examples are thiopeptide antibiotics such as thiostrepton (Jin, 2009; Lentzen et al., 2003; Nicolaou et al., 2009) (Fig. 10) or metal-chelating siderophores (Roy et al., 1999). Their metabolic stability is high, but their substitution pattern might have a stabilising/destabilising effect toward their oxidative ring scission catalysed by the cyp P450, as observed with sudoxicam (Fig. 10) (Obach et al., 2008). On the other hand, the hydrazine/hydrazides moiety (in red, Fig. 10) display a more ambiguous in vivo behaviour, most probably related to their metabolites and the potential production of radical oxydative species (ROS) and strongly reducing agents resulting from the oxydative cleavage of the hydrazine NeN bond. The formation of reactive acyl radicals has been reported for the antituberculosis isoniazid (Fig. 10), accounting also in part for the numerous side effects (Preziosi, 2007; Timmins and Deretic, 2006). The hepatotoxicity is unfortunately often observed with antidepressant hydrazides/hydrazines targeting the monoamine oxidase (MAO). Iproclozide (Fig. 10) was withdrawn from the market after reported cases of fulminant hepatitis, (Pessayre et al., 1978) and iproniazid after pronounced hepatotoxicity (Fagervall and Ross, 1986; Park et al., 2005). The least hepatotoxic isocarboxazid (Fig. 10) is readily absorbed and reaches its peak plasma concentration at 4  1 h (Koechlin et al., 1962). In addition, the carcinogenic activity of hydrazines must be closely monitored (Kean et al., 2006; Toth, 1980, 2000). Noteworthy, the above-mentioned MAO inhibition might be a valuable pleiotropic effect with these tau aggregation inhibitors. 2.3. N-Phenylamines, phenothiazines and benzothiazoles Further tau aggregation inhibitors were reported for the classes of N-phenylamines, phenothiazines and benzothiazoles (Necula et al., 2005; Pickhardt et al., 2007a, 2005a). The SAR on the Nphenylamines is close to the one described above for rhodanines and PTHs, with hydrogen bonding substituents such as nitro/ carboxylic acids on the N-phenylamines (Fig. 11) and hydrophobic binding domains provided by the aromatic rings. In contrast to the two compound classes discussed above (rhodanines and phenylthiazolyl-hydrazides), the N-phenylamines displayed far lower potencies in vitro and in cells, probably due to the non-planar conformation of the molecule. The aromatic rings connected to the nitrogen atom have indeed a complete freedom of rotation around the carbonenitrogen axis. The N-phenlyamine structure overlaps strongly with anthranilic acids used as nonsteroidal anti-inflammatory drugs (meclofenamic acid, niflumic acid). The higher cytotoxicity observed in vitro (Fig. 12) and the significant hepatotoxicity that has been reported for anthranilic acid derivatives would not recommend the use of this compound class in vivo. The phenothiazines, e.g. thionin (Fig. 11) are tricyclic structures that incorporate a sulphur and nitrogen on the central heterocycle, and are close to the above-mentioned N-phenylamine inhibitors,

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Fig. 7. Rhodanine aggregation inhibitors. IC50 and DC50 activities on tau construct K19 (Bulic et al., 2007).

sharing two aromatic rings bridged through a nitrogen atom. However, significant features distinguish phenothiazines and benzothiazoles from the N-phenylamines, mainly their cationic charge, planarity and a high level of aromatic conjugation. The planarity and aromaticity of the central heterocycle appears to be the determinant for inhibition activity on tau aggregation, since non-conjugated and distorted analogues such as perphenazine (Fig. 11) display a dramatically reduced potency (Hattori et al., 2008; Taniguchi et al., 2005). The positive charge on thionin or MTC most likely plays a secondary role since a subset of chargeneutral quinoxalines, which overlap strongly with the phenothiazines (Fig. 11), were also reported as potent tau aggregation inhibitors (Crowe et al., 2007). The highly lipophilic, aggregation inactive and charge-neutral phenothiazines have good blood brain barrier permeability as confirmed by their use as antipsychotic drugs (chlorpromazine, perphenazine). Their oral bioavailability is reported to be below 10%, mostly due to an extensive first pass metabolism targeting their aminoalkyl side chain. Noteworthy, this sensitive substitution pattern is not present on MTC and derivatives. The methylthioninium chloride compound (MTC, also known as methylene blue or Urolene) has recently been reported as

Fig. 8. Core structure of the phenylthiazolyl-hydrazides. Modifications in the flanking regions are possible at R1, R2 and R3. (Larbig et al., 2007).

a potent in vitro tau aggregation inhibitor. Preliminary results of a phase II clinical trial indicated a lower rate of decline of cognitive functions compared to placebo. MTC was orally administrated in a double blind, randomized parallel design phase II trial to 321 participants over 84 weeks. However, the blue urine staining properties of methylene blue were not matched in the placebo group. The reported results at the 50 week time point on mild to moderate cognitive impairment patients showed an ADAS-cog score decline of seven points for placebo versus one point for the MTC cohort, equivalent to a reduction of 81% of the cognitive decline rate under MTC (p < 0.0001) (Wischik et al., 2008). The results were supported by SPECT and PET brain imaging. The confirmation of these data would represent a breakthrough for the management of Alzheimer's disease and a proof of concept for the tau aggregation inhibition strategy. The pharmacokinetic

Fig. 9. Binding epitope of inhibitor BSc3094 (class of phenylthiazolyl-hydrazides) with tau construct K18 derived from STD-NMR (Pickhardt et al., 2007b). The percentages reflect the relative STD effects, representative of the binding affinities to the protein target.


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Fig. 10. Thiazole (blue) and hydrazine (red) containing structures. IC50 and DC50 for BSc3094 are for the tau construct K19 (Pickhardt et al., 2007b). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

profile of methylene blue and parent phenothiazine derivatives is abundantly documented because of their long-standing use, most prominently as antimalarial agents (Wainwright and Amaral, 2005). The ADME data analysis suggests a fairly high oral bioavailability at 72% despite its hydrophilic character (DiSanto and

Wagner, 1972a,b,c; Walter-Sack et al., 2009) due in part to the dispersion of the positive charge on the molecule by resonance. Investigations on the pharmacokinetics of methylene blue, administrated as prophylaxis of ifosfamide-associated encephalopathy indicate an half-life of 5 h, an high volume distribution and

Fig. 11. Structures of N-phenylamine-derived compounds, benzothiazoles and phenothiazines. IC50s are for aggregation inhibition of tau isoforms (Crowe et al., 2007; Khlistunova et al., 2007; Necula et al., 2005; Pickhardt et al., 2007a; Taniguchi et al., 2005).

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Fig. 12. Summary of cytotoxicity (B, black bars) and aggregation-inhibitory activity in cells (B, red bars), half-maximal concentration value for assembly-inhibition (IC50; A, green bars) and disassembly-induction (DC50; A, brown bars). Cytotoxicity assay (LDH) was performed over 24 h in the presence of 10 mM compound. For testing the inhibitory compoundactivity in cells the induced cells were treated with 15 mM compound over 5 days. The remaining aggregate-positive cells were quantified by ThS-staining. (Bulic et al., 2007; Khlistunova et al., 2007; Pickhardt et al., 2005a; Pickhardt et al., 2007b). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

significant brain penetration (Peter et al., 2000). Nevertheless the bioavailability after oral administration was found to be strongly reduced because of extensive liver accumulation, as also observed for cyanine dyes described below (Meijer et al., 1988). The long term administration of methylene blue as antimalarial agent did not induce severe side effects, as indicator of its safety (Wainwright and Amaral, 2005). Noteworthy, the pleiotropic action of methylene blue might play an important role in the observed clinical effects in AD patients, since it has been reported to also behave as an MAO inhibitor, NO-synthase inhibitor and its redox potential might rescue mitochondrial dysfunction (Atamna et al., 2008; Bruchey and Gonzalez-Lima, 2008). This last antioxidative property of methylene blue implies the presence of two chemical entities (the oxidized cationic form and the reduced neutral form also called leuco form) (Bruchey and Gonzalez-Lima, 2008) depending on the redox potential of the cellular environment (the leuco form being better compatible with the high membrane permeability observed in vivo). Despite apparent favourable pharmacokinetics, it would be of prime importance to estimate the ratios of each form in vivo, since the reduced leuco form most probably does not display anti-aggregation abilities on tau because of its obvious structural similarity with aggregation inactive phenothiazines such as perphenazine, chlorpromazine (Hattori et al., 2008; Taniguchi et al., 2005). Last but not least, benzothiazoles (Fig. 11) share with phenothiazines the characteristic positive charge and extensive aromatic conjugation. Representative example of benzothiazolebased inhibitors with excellent inhibitory potencies have been reported (N744 IC50 ¼ 300 nM, Fig. 13) (Congdon et al., 2009; Necula et al., 2005). The required moieties supporting the hydrophobic interactions commonly observed on aggregation inhibitors are provided by the benzothiazole heterocycles. This feature probably accounts for the pronounced inhibitory activity in comparison with thioflavins like ThS or ThT which are in use as reporters of the b-structure in the aggregated state, without affecting assembly as such (Necula et al., 2005). The thiocarbocyanines present a characteristic cationic charge that may interact with the target by chargeecharge interactions, but most probably only contribute to the planarity of the structure, as described above also for phenothiazines. Indeed, the planar but charge-neutral ThT analogue, Pittsburgh compound B (PiB, Fig. 13), was found to have an 45 fold increased affinity toward Ab (1e40) compared to ThT (Ki(PiB) ¼ 20 nM vs. Ki(ThT) ¼ 890 nM)

(Ikonomovic et al., 2008; Klunk et al., 2005, 2001; Mathis et al., 2002; Mathis et al., 2007). A loss of inhibitory activity with N744 has been reported at high concentration, caused by the aggregation of the compound leading to H-aggregates which consist of face-to-face columnar stacks of molecules. By contrast, the N744 inhibitor has a monomeric or dimeric structure at low concentration which is considered to be the active forms of the inhibitor (Congdon et al., 2007). A modification of the N744 compound to provide a structure that could readily form a dimer through a closed clamshell structure (compound 2, Fig. 13) ruled out the dimeric conformation as the active form of the inhibitor (Honson et al., 2007). The pharmacology of benzothiazoles is relatively well documented, owing to the use of PiB (Fig. 13) for imaging of in vivo amyloid deposition (Klunk et al., 2005; Mathis et al., 2002, 2007) and the prescription of the benzothiazole drug Riluzole (Fig. 14) for amyotrophic lateral sclerosis (NMDA receptor antagonist). Riluzole presents a satisfactory 60% bioavailabilty with a 2 h peak plasma concentration. The half-life at 12 h gives evidence of its good stability (van Kan et al., 2005). The BBB permeability has been confirmed by HPLC analysis on brain extracts in mice, however riluzole was found to be a substrate for PgP efflux transporter (Milane et al., 2007). Positively charged analogue further conjugated to heteroaromatics, also known as cyanine dyes (N744,

Fig. 13. Structures of benzothiazole aggregation inhibitors and binders.


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Fig. 14. Benzothiazole-containing drug (riluzole), tau aggregation inhibitor (C11) and parent cyanine dye (Cy3).

Fig. 13), might be prone to complexation with DNA and therefore mutagenic (Clifton and Leikin, 2003; Hilal and Taylor, 2008). Numerous cyanine dyes from this compound class family, such as Cy3 and Cy5 (Fig. 14), are extensively used in laboratories as molecular probes for nucleic acid staining. The delocalisation of the positive charge through the molecule improves their lipophilicity and thus their membrane permeability. Cyanine dyes as well as closely related positively charged redox active substances such as phentothiazines are also known as mitochondrion selective stains (Hassan and Fridovich, 1979; Johnson, 2001). Therefore, special attention must be paid during the design of this promising class of compounds in order to avoid potential toxic side effects. Promising results have been achieved with the cyanine analogue C11 (Fig. 14) on organotypic slice culture model, strongly supporting the previous in vitro observation (Congdon et al., 2009; Chang et al., 2009) A pharmacophore mapping of the anthracyclines as Ab aggregation inhibitors yielded a three point model with the aromatic rings serving as hydrophobic regions and the sugar moiety as hydrogen bond donor and acceptor (Fig. 15). Another compound class, the polyphenols, has been presented above as tau aggregation inhibitors (myrcetin, Fig. 14). These compounds show inhibitory activity on a variety of amyloids such as a-synuclein, IAPP, Ab40, PrPsc or tau (Klunk et al., 2001, 2002; Mathis et al., 2002, 2007). 2.4. Polyphenols and anthraquinones The chemical class of polyphenole is characterized by the presence of several phenols functionalities i.e. one or more hydroxyl-OH bound to aromatic rings (Fig. 15). These substances are often observed in higher plants, such as ginkgo biloba, tea bush, grape or turmeric. Polyphenols are often perceived as naturally occurring curative substances, in line with numerous claims on their therapeutical benefits linked to antibiotic, antiviral, anticancer, antidiabetic and neuroprotective activities (Han et al., 2007). However, conclusions on their in vivo effects with long term administration are risky, for the reason that pharmacological

investigations defining their bioavailability and organ distribution are often lacking. Polyphenols can be categorized according to their structure in flavonoids, phenolic acids and stilbenes (Fig. 15). Most of naturally occurring dietary polyphenols are found as glycosylated forms which are highly hydrophilic and thus poorly absorbed from the intestine. Only aglycones resulting from the hydrolysis by the intestinal flora are detected in the plasma, though their residual polarity and hydrophilicity due to the heavy decoration of the molecule with polar hydroxyls impedes their throughout passage from the intestine to the blood compartments. Their bioavailability is therefore classified as low (5e20% range) (Hu, 2007). The polyphenol fraction present in the plasma is further extensively metabolised through methylation, sulfation and glucuronidation, reducing further their bioavailability (Manach et al., 2004). The correlation with their neuroprotective effects observed in patients suffering from neurodegeneration requires therefore further investigation to clarify their bloodebrain barrier permeability and brain concentration (Ono et al., 2003; Singh et al., 2008). Further investigations should determine if the required polyphenols concentration in brain is compatible with patient safety concerns. There is indeed increasing evidence pointing at acute polyphenol toxicity at high doses, as reported for the green tea extract PolyphenonÒ (Kapetanovic et al., 2009; Mennen et al., 2005). A prominent example of the ambiguous properties of polyphenols is documented for phytoestogen isoflavones whose similarity with oestrogens might confer a carcinogenic oestrogenic activity (Allred et al., 2004; Anupongsanugool et al., 2005; Dixon, 2004). Numerous polyphenols show inhibitory activity on a variety of amyloids such as a-synuclein, IAPP, Ab40, PrPsc. Myrcetin has been reported as tau aggregation inhibitors with a 1.2 mM IC50 and the in vitro data indicate that they interfere with the elongation phase of fibril assembly (Howlett et al., 1999; Porat et al., 2006; Taniguchi et al., 2005; Yang et al., 2005). The structure activity-relationship for polyphenolic aggregation inhibitors on tau and Ab points toward an essential role of the hydroxyls on the phenolic moiety, (Ono et al., 2003) since ether analogues displayed reduced activities. Phenolic hydroxyls are known for their greater acidity than aliphatic hydroxyls, and for their ability to form hydrogen bonds. The requirement for hydrogen bonding was also observed for scyllo-inositol inhibitors (McLaurin et al., 2000). Symmetrical structures such those of rosmarinic acid and curcumin and an optimal molecular length of 16e19 Å is reported to be most favourable on Ab40 aggregation inhibition (Reinke and Gestwicki, 2007). Notewothy, the planarity of the structure appears to be of high importance, as also described above for other aggregation inhibitors, deduced from the comparative aggregation-inhibitory potencies between the planar cyanidin and the non-planar and inactive catechin (Fig. 16) (Hattori et al., 2008; Taniguchi et al., 2005). Anthracyclines are not classified as polyphenols, yet are close analogues which also display aggregation inhibition abilities on tau and Ab40 (Howlett et al., 1999; Taniguchi et al., 2005). The

Fig. 15. Classification of polyphenols.

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anthraquinones all share a tricyclic structure with one or more phenolic moieties (Fig. 17) also observed in polyphenols as described above. The anthracyclines are known as intercalating cytostatics that are successfully employed in anticancer therapies, but for the same reason they present a hazardous toxicological profile for long term administration in AD. The side effects with adriamycin (Fig. 17) are well-known, including alopecia and congestive heart failure. The formation of reactive oxygen species (ROS) is well documented, resulting from adriamycin iron chelation (Xu et al., 2005). Our LDH cytotoxicity data (Fig. 12) confirm substantial toxicity in vitro. The glycones anthracyclines are generally administrated intravenously, because of their low bioavailability due to the high hydrophylicity of the daunosamine sugar (blue on ring D, Fig. 11). The agylcones, such as emodin (Fig. 17), have a better but insufficient bioavailability (22.5%) due to the high residual hydrophilicity (Teng et al., 2007). The ingestion of anthraquinones is one of the major current self-medications, due to the laxative properties of the compounds (Mueller et al., 1999; van Gorkom et al., 1999). The over-the-counter herbal extracts contain principally emodins (Fig. 17) and sennosides which exert their laxative effects by damaging colonic epithelial cells. Their use has been correlated with a higher incidence of colon cancer and others (Mueller et al., 1998; van Gorkom et al., 1999). The five compounds depicted on Fig. 17 were able to inhibit the aggregation of the K19 tau construct and also induced the disaggregation of preformed aggregates, with IC50 values between 1.1 mM and 2.4 mM and DC50 values between 2.2 mM and 3.8 mM (Pickhardt et al., 2005a). One exception is compound PHF005 which showed a markedly lower activity, probably due to the flexibility of the structure compared to closed tricyclic structures (rotation axis depicted by the arrow in Fig. 17). Moreover, the substitutions on the ring A (daunorubicin, Fig. 17) did not appear to play a crucial role, since different patterns yielded similar inhibitory potencies. Noteworthy, the ring D in daunorubicin and adriamycin bearing the sugar does not give a competitive advantage compared to PHF016, indicating that the compounds are only moderately sensitive to the substitutions on that ring and accommodate even bulky substitutions such as sugars. A pharmacophore mapping of the anthracyclines as Ab aggregation inhibitors yielded a three point model with the aromatic rings serving as hydrophobic regions and the sugar moiety as hydrogen bond donor and acceptor (Howlett et al., 1999).


3. Binding mode Folding pathways are complex and started from non-toxic native monomeric peptides to fibrils implying the presence of various potential targets for aggregation inhibitors and disaggregators (Howlett, 2001; LeVine, 2002). The numerous questions about the mechanisms and targets are still awaiting answers. As reported for Ab, it probably involves the trapping of the monomer or an assembly intermediate involved in the dynamic equilibrium between fibril and monomer (Harper and Lansbury, 1997; Matsuoka et al., 2003; von Bergen et al., 2005). The compounds may also be able to interact directly with the aggregated structure and disturb the proteineprotein p-stacking arrangement of the fibrils. A different binding mode and different targets for inhibition and disaggregation on the complex pathway of aggregation from the native non-toxic peptide to fibrils could therefore rationalize the differences in potencies between observed IC50s and DC50s. The binding epitopes obtained by STD-NMR between a PTH inhibitor and a monomeric tau peptide depicted in Fig. 9 illustrate that the direct interaction of small molecules with the monomeric peptide might be a common feature among aggregation inhibitors and disaggregation promoters. However, binders from the thioflavin family such as ThT have been reported to only bind to pre-aggregates and aggregate forms, showing that multiple binding modes are likely (LeVine, 1993). Noteworthy, several compounds listed above as aggregation inhibitors have been observed to form also unordered micellar structures inherent to their hydrophobic nature, and are therefore suspected to be unspecific through colloidal inhibition (Feng et al., 2008). 4. Summary and outlook A close interplay between Ab-amyloids and tau aggregation is emerging, long after the histopathological observations on postmortem brains pointing at an intimate correlation between the load of neurofibrillary tangles within neurons and cellular degeneration (King et al., 2006; Park and Ferreira, 2005; Rapoport et al., 2002; Roberson et al., 2007; Small and Duff, 2008; Talaga and Quere, 2002). Even though the elucidation of the cascade of events leading to tau aggregation is not yet achieved, several lines of evidence suggest that Ab oligomers or aggregates, commonly considered as the major culprits of AD, might also exert their toxic

Fig. 16. Planar (cyanidin) and non-planar (catechin) polyphenols. IC50s for tau aggregation inhibition (Hattori et al., 2008; Taniguchi et al., 2005).


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The ability to form H- or J-aggregates by many of the aggregation inhibitors might indicate a specific binding mode involving the self-assembly of the compounds to form a supramolecular structure (Piekarska et al., 1996; Skowronek et al., 2000; Spolnik et al., 2007; Von Berlepsch et al., 2000) distinct from the unordered micelles typically observed with promiscuous inhibitors (Feng et al., 2008; McGovern et al., 2003). The determination of the active form of the inhibitors and their rational design will present an exciting challenge to reach potential drug candidates. Acknowledgements

Fig. 17. Structures of anthraquinone-derived compounds. The glycoside parts are depicted in blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

function via tau as a constitutive intracellular component (King et al., 2006; Park and Ferreira, 2005; Rapoport et al., 2002; Roberson et al., 2007; Talaga and Quere, 2002). Oligomeric tau can also play a role in the pathogenic mechanism of AD, possibly even via extracellular pathways (Frost et al., 2009; Clavaguera et al., 2009), and putative inhibitors must be testet with regard of their ability to inhibit possible oligomer formation. Our own observations using a charge-neutral compound of moderate in vitro activity such as 14 (Fig. 7) allowed the study of the effects of tau filament disassembly on cellular viability, which resulted in the prevention or reversal of the toxicity caused by tau aggregation in the cytosol (Khlistunova et al., 2007; Meyer-Luehmann et al., 2008; Winklhofer et al., 2008). Therefore, apart from providing a reliable biomarker for Alzheimer's disease diagnostic, tau aggregation inhibitors and disaggregators might complement the current therapeutic approaches, as suggested by the recent clinical trials with methylene blue and the mitigated results obtained after Abimmunisation alone (Holmes et al., 2008; Wischik et al., 1996, 2007). In view of the neuronal toxicity mediated by small oligomers (Lashuel and Grillo-Bosch, 2005; Necula et al., 2007), it has been recently suggested that interventions that reduce amyloid load but increase small oligomers could be harmful (Cheng et al., 2007). In principle, it is therefore necessary to determine the binding properties of the amyloid inhibitor compounds to the small oligomeric amyloid precursors. Noteworthy, recent findings indicate that several Ab-amyloid binders and inhibitors also bind to Ab oligomers and have neuroprotective effects (Hong et al., 2007; Maezawa et al., 2008). Shared structural features between the compound classes start to emerge, with the presence of aromatic/hydrophobic patches and hydrogen bonding elements along flat extended structures. The development of improved aggregation inhibitors will be linked to the understanding of their binding mode at the molecular level, and the ability to integrate crucial elements of pharmacology at an early stage of the compounds development to address the in vivo toxicity, brain permeability and plasma stability. Especially the negative or positive charges shared by most aggregation inhibitors impede membrane permeability and therefore their use in vivo. The successful compound optimization toward charge-neutral cell permeable structures is illustrated by the design of imaging agents for diagnosis such as Pittsburgh compound B (PIB) or methoxy-X04, inspired by the poorly permeable thioflavins or Congo red analogues (Cai et al., 2007; Kinosian et al., 2000; Klunk et al., 2001, 2005, 2002, 1989; Mathis et al., 2007).

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