StructuralDeterminantsofTauAggregationInhibitor Potency · property that describes how easily...

Structural Determinants of Tau Aggregation InhibitorPotency*

Received for publication, July 21, 2013, and in revised form, September 16, 2013 Published, JBC Papers in Press, September 26, 2013, DOI 10.1074/jbc.M113.503474

Kelsey N. Schafer1, Katryna Cisek1, Carol J. Huseby, Edward Chang, and Jeff Kuret2

From the Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University,Columbus, Ohio 43210

Background: Mechanistic insight into small-molecule Tau aggregation inhibitors is needed for their advancement astherapeutic agents.Results: Structure-activity relationship analysis identified polarizability as a common descriptor of inhibitor potency.Conclusion: Flat, highly polarizable ligands stabilize soluble oligomeric complexes at the expense of filamentous aggregates.Significance: The findings suggest a basis for rational improvement of ligand potency, whereas maintaining bioavailability.

Small-molecule Tau aggregation inhibitors are under investi-gation as potential therapeutic agents against Alzheimer dis-ease. Many such inhibitors have been identified in vitro, buttheir potency-driving features, and their molecular targets inthe Tau aggregation pathway, have resisted identification. Pre-viously we proposed ligand polarizability, a measure of electrondelocalization, as a candidate descriptor of inhibitor potency.Here we tested this hypothesis by correlating the ground statepolarizabilities of cyanine, phenothiazine, and arylmethinederivatives calculated using ab initio quantum methods withinhibitory potency values determined in the presence of octa-decyl sulfate inducer under reducing conditions. A series of rho-danine analogs was analyzed as well using potency values dis-closed in the literature. Results showed that polarizability andinhibitory potency directly correlated within all four series. Toidentify putative binding targets, representativemembers of thefour chemotypes were added to aggregation reactions, wherethey were found to stabilize soluble, but SDS-resistant Tau spe-cies at the expense of filamentous aggregates. Using SDS resis-tance as a secondary assay, and a library of Tau deletion andmissensemutants as targets, interaction with cyanine was local-ized to the microtubule binding repeat region. Moreover, theSDS-resistant phenotype was completely dependent on thepresence of octadecyl sulfate inducer, but not intact PHF6/PH6*hexapeptide motifs, indicating that cyanine interacted with aspecies in the aggregation pathway prior to nucleus formation.Together the data suggest that flat, highly polarizable ligandsinhibit Tau aggregation by interacting with folded species in theaggregation pathway and driving their assembly into soluble buthighly stable Tau oligomers.

Because the appearance of Tau protein-bearing lesions inAlzheimer disease (AD)3 correlates with neurodegenerationand cognitive decline (1, 2), various approaches for inhibitingtheir formation are under investigation as potential therapiesagainst disease progression. An attractive target is the Tauaggregation reaction itself, which is closely associated withlesion formation but not normal Tau function (3). Despite hav-ing the advantage of disease specificity, the approach of directlyinhibiting Tau protein-protein interactions faces hurdles,including the lack of a distinguishable “binding pocket” on Taumonomer because of its natively unfolded structure, and thelarge surface areas that mediate Tau-Tau interactions, whichcould require impractically large molecules for effective antag-onism (4). Under such conditions, Tau aggregation inhibitorswould be expected to lack adequate binding affinity and brainbioavailability for therapeutic utility.Nonetheless, small-molecule Tau aggregation inhibitors

have been reported in the literature (5–10). These consist ofvarious chemotypes, including but not limited to phenothia-zines (6), polyphenols (7), porphyrins (7), rhodanines (8), andcyanines (9, 10), with phenothiazine derivative methylene blueshowing promise for delaying progression of AD (11). Eachscaffold family differs inmolecular weight, hydrophobicity, andsterics, yet all inhibit Tau aggregation in vitro at micromolar oreven submicromolar concentrations. These findings suggestthat inhibition of Tau aggregation with small molecules is fea-sible, and raise the question of how inhibitory efficacy andpotency is achieved across scaffold classes. The problem is sig-nificant because most inhibitors identified to date on the basisof in vitro approaches have physical-chemical characteristicsinconsistent with efficient blood-brain barrier penetration,hindering their preclinical evaluation. For example, thiacarbo-cyanine, phenothiazine, and arylmethine inhibitors are perma-nent cations (6, 10, 12), whereas porphyrin and pthalocyaninederivatives have relatively high molecular masses (7). Unlikethese molecules, aggregation antagonists with improved bio-availability could have therapeutic utility.

* This work was supported, in whole or in part, by National Institutes of HealthGrant AG14452, Alzheimer’s Disease Drug Discovery Foundation Grant281205, and an allocation of computing time from the Ohio Supercom-puter Center (PAS0453).

1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: 1060 Carmack Rd., Colum-

bus, OH 43210. Tel.: 614-688-5894; Fax: 614-292-5379; E-mail: [email protected].

3 The abbreviations used are: AD, Alzheimer disease; ODS, octadecyl sulfate;MTBR, microtubule binding repeat; PLR, partial least squares regression;SAR, structure-activity relationship; DMSO, dimethyl sulfoxide.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 45, pp. 32599 –32611, November 8, 2013© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

NOVEMBER 8, 2013 • VOLUME 288 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 32599

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Descriptors of inhibitor potency traditionally have beenidentified through structure-activity relationship (SAR) analy-sis, where compound structure is systematically varied and cor-related with biological activity. Using this approach, we postu-lated that ligand polarizability was a candidate descriptor ofcyanine potency in vitro (10, 13). Polarizability is an electronicproperty that describes how easily electron density can shiftabout a molecule when exposed to an external electric field,such as an adjacent dipole or ion. For planar molecules, highpolarizability can support strong van der Waals interactionswith flat surfaces exposed on binding partners (14). In contrast,reports of SAR analysis within rhodanine and phenothiazinefamilies have focused on sterics rather than chemical descrip-tors of graded biological potency (15, 16). As a result, the activ-ity driving commonalities among diverse chemotypes haveremained elusive.Full understanding of the mechanism of Tau aggregation

inhibition will require perspective from the point of view of thetarget (i.e. the Tau species with which the inhibitor interacts) aswell as from that of compound (i.e. the descriptors of inhibitorypotency). In vitro, the aggregation pathway beginswith the con-version of natively unfoldedTaumonomer into an aggregation-prone confirmation (17). This step can be accelerated in vitrobyinclusion of an “inducer” such as heparin or anionic detergent(reviewed in Ref. 18). Once aggregation competent conforma-tions are adopted, the rate-limiting step toward fibrillizationbecomes nucleation. This is followed by extension, where Taumonomers add onto filament ends (17). Tau aggregation inhib-itors have been reported to inhibit the forward reaction as wellas drive disaggregation ofmature filaments, but theirmoleculartargets have been identified in only a few cases. For example,aldehydes can covalently modify Tau protein monomer,thereby trapping it in aggregation incompetent forms (19).Other compounds promote Cys oxidation under non-reducingconditions and extended incubation times, again yieldingassembly incompetent monomer (20, 21). Neither of thesecovalent mechanisms is predicted to have utility in vivo. How-ever, other protein aggregation inhibitors are active in the pres-ence of thiol reducing agents or are otherwise not associatedwith protein oxidation or alkylation. For example, curcuminhas been reported to increase the reconfiguration rate of �-synuclein, such that occupancy of assembly competent confor-mations is minimized (22, 23). This mechanism implies directbut transient interaction between inhibitor and nativelyunfolded proteinmonomer. Finally, certain aromatic heterocy-cles have been reported to trap Tau in the form of soluble oligo-meric species (7), even when tested under reducing conditions(24). Similarly, in a study of �-synuclein aggregation, polyphe-nol, phenothiazine, polyene macrolide, porphyrin, and Congored derivatives were found to stabilize SDS- and Sarkosyl-insol-uble oligomer as observed by SDS-PAGE analysis (25). How-ever, neither the target of ligand binding nor the descriptors ofinhibitor affinity were identified in these studies.To clarify the mechanism of inhibitory action from the per-

spectives of both compound and protein, here we investigatethe activity of four series of Tau aggregation inhibitors, com-posed of cyanine, phenothiazine, arylmethine, and rhodaninederivatives under reducing conditions. The results point

toward ligand polarizability as a common descriptor of inhibi-tory potency, and at least partially folded Tau intermediates astheir molecular target.

EXPERIMENTAL PROCEDURES

Materials—Recombinant His-tagged full-length wild-typeTau isoforms (2N4R and 0N3R), Tau truncation mutants(2N4R�376, 2N4R�344, 2N4R�314, 2N4R�252, and 2N4R252–376),Tau missense mutants (2N4RI277P,I308P and 2N4RC291A,C322A),and tauopathy missense mutants (2N4RR5L, 2N4RG272V,2N4RP301L, 2N4RV337M, and 2N4RR406W), as well as non-His-tagged 2N4R Tau (2N4R�6His) were prepared as described pre-viously (26–30). These preparations were �80% pure on thebasis of SDS-PAGE (Coomassie Blue stain).Mousemonoclonalantibody Tau5 (31) was a gift from L.I. Binder (NorthwesternUniversity, IL).Horseradish peroxidase-linked goat anti-mouseimmunoglobulin G (IgG) was from Kirkegaard and Perry Lab-oratories (Gaithersburg, MD). Nitrocellulose membrane (0.2-�m porosity) was from Bio-Rad. Formvar/carbon-coated cop-per grids, glutaraldehyde, and uranyl acetate were obtainedfrom Electron Microscopy Sciences (Fort Washington, PA).Aggregation inducer ODS was obtained from Lancaster Syn-thesis (Pelham, NH) and dissolved in 1:1 water:isopropyl alco-hol before use. Compounds tested in vitro in this study includedcyanine 1 (Sigma) and macrocyclic cyanine 9 (prepared as inRef. 13), phenothiazines 10 (Acros Organics,Morris Plains, NJ)and 11–14 (Sigma), arylmethines 15–19 (Sigma), and rhoda-nine 28A (ChemBridge). Compoundswere at least 95% pure onthe basis of high performance liquid chromatography or thin-layer chromatography analysis and were dissolved in DMSOprior to use.Tau Aggregation—Recombinant human Tau preparations (3

�M) were incubated (37 °C) without agitation in assemblybuffer (10 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM dithiothre-itol) for up to 24 h in the presence or absence of fibrillizationinducer ODS (50 �M) and inhibitors (up to 10 �M). Controlreactions contained DMSO vehicle, which was limited to 1%(v/v) final concentration in all aggregation reactions. Followingincubation, reactions were immediately assayed by either elec-tron microscopy or SDS-PAGE, or fractionated by sedimenta-tion as described below.Electron Microscopy—Reaction aliquots (50 �l final volume)

were treatedwith 2% glutaraldehyde (final concentration), thenmounted on grids and negatively stainedwith 2% uranyl acetateas described previously (32). Random fields were viewed with aTecnai G2 Spirit BioTWIN transmission electron microscope(FEI, Hillsboro, OR) operated at 80 kV and �23,000–49,000magnification. Total filament length is defined as the sumof thelengths of all resolved filaments per field and is reported � S.D.SDS-PAGE—Samples were boiled for 2 min in the presence

of sample buffer under reducing conditions (3.75% 2-mercapto-ethanol), thenapplied topolyacrylamide slabgels asdescribedpre-viously (33).Sedimentation—To separate soluble from insoluble Tau spe-

cies, aliquots of aggregation reactions (100 �l final volume)were centrifuged (100,000 � g) in a Ti 42.2 rotor for 30 min at4 °C (34). Pellets (P1 fraction) were re-suspended in 100 �l ofassembly buffer by vigorous trituration, whereas supernatants

Tau Aggregation Inhibitor Structure and Mechanism

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(S1 fraction) were removed and subjected to density gradientsedimentation to further fractionate soluble Tau species.Sucrose step gradients composed of 20, 30, 40, and 50% sucroselayers (1 ml/layer) prepared in assembly buffer (35, 36) werecentrifuged (100,000� g) in an SW55 rotor for 2 h at 4 °C. Eachlayer was then collected and analyzed for Tau content asdescribed below.Tau Protein Assay—The Tau content of fractions was deter-

mined by dot blot analysis using nitrocellulose membranes (0.2�m porosity) as described previously (37). The membraneswere blocked in 5% nonfat drymilk dissolved in blocking buffer(100 mM Tris-HCl, pH 7.4, and 150 mM NaCl) for 1 h and thenincubated with primary antibody Tau5 at 1:5000 dilution for2 h. Membranes were then washed three times with blockingbuffer and incubated with horseradish peroxidase-linked sec-ondary antibody for 2 h. Afterwashing another three timeswithblocking buffer, membranes were imaged using the EnhancedChemiluminescence Western blotting Analysis System (GEHealthcare, Buckinghamshire, UK) recorded on an Omega12iC Molecular Imaging System and quantified using Ultra-Quant software (UltraLum, Claremont, CA, USA).Spectrophotometry—Absorbance measurements were made

in neatmethanol solvent as described previously (10). Themax-imum absorbance wavelength (�max) was determined from fitsof the data to a multi-Gaussian function (38).Computational Chemistry—Semi-empirical descriptors were

calculated for each analyzed compoundusingE-DRAGON1.0, anonline implementation of theDRAGON5.4molecular descrip-tor generator (39) that computes �1600 descriptors catego-rized into 20 logical blocks (40). The starting E-DRAGONdescriptor setwas then pruned on the basis of reported dye SARstudies (41, 42) to yield a focused molecular property set com-prising 278 descriptors representing five logical blocks: 48 con-stitutional descriptors, 33 connectivity indices, 154 functionalgroup counts, 14 charge descriptors, and 29 molecular proper-ties. The pruned descriptor set was then augmented with clogPand topological polar surface area values were calculated usingthe Molinspiration Property Calculation Service, and withpolarizability (�) values calculated at the quantum level usingdensity functional theorymethods implemented inGaussian 09(G09) (43). We described these methods in detail previously(14, 37). Quantum calculations were performed in implicit sol-vent using the density field theory functional B3LYP and the6–311��G(d,p) basis set to more accurately model the pho-tophysical properties of dye molecules (44).To correlate descriptor and IC50 data, Genetic algorithm-

PLR analysis was performed using the Virtual ComputationalChemistry Laboratory, an online portal for computationalchemistry tools (last accessed 1 June 2013). PLR models wereoptimized on the basis of leave-one-out cross-validation(Q2

loo). External validation of PLR models was performed in Rversion 2.13.0. The test set was chosen using the Kennard-Stone algorithm (as implemented in the ken.sto function ofthe soil.spec package). Statistical analysis was performed asdescribed previously (45), whereR02,R�0

2, k, and k� correspond tothe correlation coefficients and slopes of linear regressions con-strained through the origin (46).

Data Analysis—Concentration-effect data from either filteror electronmicroscopy assays were normalized to DMSO vehi-cle control reactions and fit to the function,

y � ymin �ymax � ymin

�1 � 10�log IC50 � log x�n�(Eq. 1)

where y and ymax represent theminimum andmaximum aggre-gation measured in the presence and absence of inhibitor (atconcentration x), respectively, n is the Hill coefficient, and IC50is the concentration of inhibitor that results in 50% of maximalinhibition. IC50 values are reported � S.E. of the estimate.

All measured parameters are reported as mean � S.D. ofbiological replicates. Differences between groups were ana-lyzed by one-way analysis of variance and Tukey’s post hocmultiple comparison test.

RESULTS

Structure Activity Relationship Analysis of Cationic TauAggregation Inhibitors—To test whether polarizability was adescriptor of Tau aggregation inhibitory potency, experimen-tation focused initially on cyanine, phenothiazine, and arylme-thine inhibitor families. These series were chosen because rep-resentative members were commercially available that variednarrowly in size and sterics, but more widely in potentialdescriptors of inhibitor activity such as polarizability. The cya-nine series included eightmolecules (1–8) with varying hetero-cycle and substituent composition, and one containing twothiacarbocyanine moieties in macrocyclic configuration (9)(Table 1). The inhibitory potencies of cyanines 1–8 againstODS-induced aggregation of full-length recombinant human2N4R Tau under near-physiological conditions of Tau concen-tration, pH, ionic strength, and reducing conditions werereported previously (10), whereas the concentration-effectrelationship for 9 assayed under identical conditions is shownin Fig. 1a. ODS detergent was used as aggregation inducer inthese experiments because of its efficacy with full-length Tauisoforms (47) and because of the reported ability of micellardetergents to depress small-molecule aggregation associatedwith promiscuous activity (48). Overall, the nine cyanines werefound to vary in IC50 value over nearly 2 orders of magnitude�10 �M (Table 1). When these values were compared against

TABLE 1Cyanine structures and properties

a Values for 1–8 taken from (10).b Macrocylic cyanine (13).




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polarizability (�) calculated for the ground state using ab initioquantummethods (Table 1), a strong log-linear correlationwasobserved (R2 0.72; Fig. 2a). In contrast, only weak correla-tions were observed between IC50 and clogP, topological polarsurface area, orMr (shown for clogP only in Fig. 2b). These dataindicate that increasing electron delocalization within theground-state cyanine ring system correlated positively withaggregation inhibitory potency within this narrow series.The approach was then extended to phenothiazine and aryl-

methine derivatives. The phenothiazines included the estab-lished aggregation inhibitor 10 (methylene blue) and four ana-

logs (11–14) that differed solely in the strengths of the electrondonor/acceptor groups flanking the phenothiazine nucleus(Table 2). As a result, this series varied over relatively narrowranges of molecular weight and hydrophobicity (Table 2).When assayed under identical conditions as the cyanine inhib-itors described above, only the two phenothiazines with great-est � value inhibited Tau aggregation with IC50 � 10 �M (Fig.1b). Similarly, the arylmethine series comprised four triarylme-thines that varied solely in the composition of electron donor/acceptor groups (15–18) and Bindschedler’s green (19) (Table2). When assayed under identical conditions as the cyanineinhibitors described above, only the three arylmethines withgreatest � inhibited Tau aggregation with IC50 � 10 �M (Fig.1c). Within the phenothiazine and triarylmethine series, therank order of potency paralleled the strengths of constituentelectron donor moieties (-N(CH3)2 � -NHCH3 � -NH2 �

FIGURE 1. Small molecule-mediated inhibition of Tau aggregation. Full-length recombinant Tau (3 �M) was incubated with aggregation inducer ODS (50�M) without agitation (16 h, 37 °C) in the presence of a, cyanine 9; b, phenothiazines 11-14; c, arylmethines 15-19; or DMSO vehicle alone, then assayed forfilament formation by electron microscopy. Each data point represents aggregation expressed as a normalized percentage of filament formation measured inthe presence of DMSO vehicle alone (triplicate determination � S.D.), whereas each solid line represents best fit of the data points to Equation 1. IC50 valuescalculated from these data are reported in Table 1.

FIGURE 2. Tau aggregation inhibitor potency correlates with ligandpolarizability. IC50 values reported for cyanines 1-9 (Table 1) (10) were plot-ted as a function of a, polarizability and b, clogP values calculated by ab initioand semi-empirical methods, respectively. Solid lines represent best fit of thedata to linear regression, with the resultant correlation coefficient reported asR2. Cyanine inhibitory potency correlated directly with polarizability but notclogP.

TABLE 2Phenothiazine and arylmethine structures and properties

a Value for 10 taken from (Ref. 10). ND, not determined.




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-OH) established on the basis of their Hammett substituentconstants (49). Overall, these data implicate polarizability asone descriptor of inhibitory potency in three distinct scaffolds,

and identify electron donating substituents as being effectivedrivers of ligand polarizability.Structure-Activity Relationship Analysis of Rhodanine Aggre-

gation Inhibitors—Cyanines, phenothiazines, and arylmethinesall share permanent cationic character, which may limit theirability to cross the blood-brain barrier. In contrast, ideal Tauaggregation inhibitors would likely have neutral charge toenhance brain penetrability. To test whether polarizabilitycould modulate inhibitory activity in a neutral chemotype, astructure-activity relationship analysis of rhodanine deriva-tives was performed. IC50 values for these compounds, whichspanned nearly 4 orders of magnitude, were taken from theliterature (8). However, because of the computational chal-lenges raised by adamantyl, boronyl, and ferrocenyl moieties,only 45 of the reported 52 molecules were analyzed herein(Table 3). Many members of this library were predicted to becharged at physiological pH, but this resulted from ionizablependent R groups rather than from the core scaffold itself(which was neutral).Because of the moderate size of this dataset, and its substan-

tial structural diversity, a cheminformatics approach was takento identify the best combination of descriptors for predictinginhibitory activity.On the basis of a training set of 39molecules,the best PLRmodel rationalized pIC50 (i.e. -log IC50) in terms of35 molecular descriptors (x variables) collapsed into six linearcombinations (latent t variables) (Table 4). On the basis ofinternal leave-one-out cross-validation (Q2

loo (50)) the correla-tion was adequately strong (Table 4). Moreover, when themodelwas applied to an external test set (i.e. six compounds notused in the calibration), the resulting correlation between pre-dicted and observed IC50 values met target criteria of slope andgoodness of fit (46) necessary for predictive utility (Table 5,Fig. 3). Together, the internal and external validation experi-ments indicated that an acceptable rationalization of rhodanineactivity over a broad concentration range was achieved.The five highest-weighted and therefore top-ranked descrip-

tors identified by the model as contributing to rhodaninepotency were AlogS, �, BLTF96, AlogP, and topological polarsurface area (Table 4). The second highest weighted of thesewas �, and its positive coefficient indicated that increasingpolarizability correlated directly with rhodanine potency. Thisresult was consistent with the cyanine, phenothiazine, and aryl-methine series described above. The next three descriptors

TABLE 3Rhodanine structures and properties

TABLE 3Continued

a Values taken from Ref. 8.




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related to compound hydrophobicity. That inhibitory potencycorrelated directly with AlogP (51) and inversely with topolog-ical polar surface area (52), suggested that compound hydro-phobicity also contributed to rhodanine activity. However, thepositive correlation with BLTF96, another hydrophobicityindex that varied inversely with octanol-water partition coeffi-cient (53), indicated that the contribution of hydrophobicitymay be complex and/or have an optimum. In fact, the positivecorrelation between the highest weighted descriptor, AlogS(54), and potency revealed that for this series, which containeda number of large hydrophobic analogs, maintenance of aque-ous solubility was of paramount importance. Taken together,this analysis confirmed polarizability as an important molecu-lar descriptor for Tau aggregation inhibitor potency that wasshared among multiple chemotypes.Aggregation Inhibitors Stabilize Soluble TauOligomers—The

foregoing identified flat, highly polarizable compounds asmedi-ators of Tau aggregation antagonist activity. To gain insight intotheir mechanism of action, the products of recombinant human2N4R Tau aggregation reactions performed in the presence andabsence of themost potent commercially available inhibitor fromeach compound family (cyanine 1, phenothiazine 10, triarylme-thine 15, and rhodanine 28A) were separated by sedimentationand quantified by immunoblot analysis. In the absence of ODSinducer, Tau protein did not aggregate, and so nearly all Tauremained in the supernatant fraction (i.e. the S1 fraction) aftercentrifugation (Fig. 4a). In the presence of ODS, however, Tauaggregated to form filaments as reported previously (47), and themajority of protein product migrated with the insoluble fraction(Fig. 4a). In contrast, whenODS and1,10, or15were present, thedistribution of reaction products shifted toward the soluble frac-tion (p 0.001; Fig. 4a). Rhodanine 28Awas the least efficaciousinhibitor under these conditions, but it too shifted reaction prod-ucts to the soluble fraction (p0.05;Fig. 4a).Thesedata showthatall four inhibitors acted to stabilize soluble forms of Tau proteinwhen present during the aggregation reaction.To characterize the Tau species stabilized by inhibitors, S1

supernatants from each experiment were subfractionated ondiscontinuous gradients containing 20–50% sucrose. Theseconditions were chosen because they had been reported toresolveTaumonomer, which remainsmostly in the lowest den-sity sucrose layer, from soluble oligomeric Tau complexes,which appear mostly in denser sucrose layers (36). Indeed,when the supernatant from the control Tau reaction lackinginducer was fractionated on the gradient, the overwhelmingmajority of immunoreactivity migrated within the least denselayer (20% sucrose; Fig. 4b). In contrast, soluble products result-ing from the aggregation control prepared in the presence ofODS migrated predominantly in the denser layers, consistentwith the formation of small soluble aggregates (Fig. 4b). Thepresence of aggregation inhibitors increased the levels of Taumigrating in the denser fractions still further. In particular, Taulevels in the 40% sucrose layer were significantly elevated for 1,10, and 15 (Fig. 4b). In contrast, the differences in Tau levelsproduced by 28A reached statistical significance only in the20% sucrose layer (Fig. 4b). Together, the sedimentation datawere consistent with Tau aggregation inhibitors acting to sta-bilize soluble Tau species of varying density.

FIGURE 3. Correlation plots for PLR models of rhodanine aggregationinhibitors. Each point represents observed versus predicted log IC50 valuesfor training and test sets of 39 and 6 compounds, respectively, whereas thelines represent linear regression of the data points (solid line, training set;dashed line, test set). As indicated by R2 values, correlations betweenobserved and predicted log IC50 values were adequately strong for both train-ing and test sets.

TABLE 4PLR model statistics

a Ranked by absolute value.

TABLE 5PLR external validation

a Target values from Golbraikh and Tropsha (46).




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To further characterize soluble Tau species, aliquots of eachS1 fraction were analyzed by SDS-PAGE after boiling underreducing conditions. In the absence of inhibitor, filamentousTau aggregates were efficiently solubilized in SDS/2-mercapto-ethanol-containing sample buffer to yield monomer migratingat �67 kDa (Fig. 5). In contrast, the presence of inhibitorsdepressed the amount of Tau migrating in the monomer posi-tion, with 1, 10, and 28A reaching statistical significance underthese conditions (Fig. 5). These data suggested that the solubleTau species stabilized by aggregation inhibitors resisted SDSsolubilization. To further characterize this behavior, the timecourse of cyanine 1-mediated effects of Tau migration in SDS-PAGE was investigated. Compared with aggregation reactionscontaining DMSO vehicle alone, incubation in the presence of 1led to decreasing levels of solubilized monomer and increasinglevels of slowly migrating species within 1 h (Fig. 6). After 2 hincubation with 1, SDS-soluble monomer decreased to 25% ofcontrol levels, whereas slowlymigrating species appeared that didnotenter thegel (Fig.6).Aftera4-h incubation, themajorityofTauwas renderedSDS-insoluble (Fig. 6). Togetherwith sedimentationdata, these findings indicate that cyanine 1 and Tau interact rap-idly to form highly stable oligomeric complexes that resist solubi-lization in SDS under reducing conditions.Cyanine-mediated Oligomer Formation Requires Inducer

and the Tau MTBR Region—Because Tau oligomers were sta-bilizedmost strongly by cyanine 1, this compound was selectedas a probe for Tau-ligand interactions.Moreover, because of itsspeed and simplicity, loss of SDS solubility was used as a sec-ondary assay for quantifying interactions between 1 and a rangeof Tau constructs thatwould be difficult to capture using aggre-gation assays alone. To identify the regions of Tau protein thatmediate inhibitor activity, the ability of 1 to depress SDS solu-bility of Tau truncation mutants was investigated. Because allTau constructs used to characterize inhibitor activity up to thispoint were tagged with an N-terminal His tag, we first com-pared the performance of recombinant wild-type 2N4R and2N4R�6His proteins in the presence or absence of compound 1(see Fig. 7a for maps of 2N4R Tau and the truncation mutants

used herein). As before, incubation of 2N4R Tau protein in thepresence of 1 under aggregation conditions lowered its SDSsolubility, and the absence of the His tag did not change this

FIGURE 4. Tau aggregation inhibitors stabilize soluble oligomeric forms of Tau protein. The products of Tau aggregation reactions (16 h, 37 °C) conducted in thepresence or absence of ODS inducer and cyanine 1, phenothiazine 10, arylmethine 15, rhodanine 28A, or DMSO vehicle alone were centrifuged (100,000 � g for 30min). Levels of total Tau protein in supernatant (S) and pellet (P) fractions were then quantified by dot blot analysis using monoclonal antibody Tau5. a, quantificationof Tau in each fraction (n 3; reported � S.D.), normalized to the negative control for aggregation (no ODS inducer, no inhibitor). The presence of ODS-inducedaggregation, and shifted products to the pellet fraction (white bars) is shown. In contrast, the presence of aggregation inhibitors shifted the reaction products backtoward the supernatant fractions. *, p 0.1; **, p 0.01; and ***, p 0.001, for comparison of supernatant fractions to positive aggregation control; #, p 0.05;###, p 0.001, for comparison of pellet fractions against the positive aggregation control. b, the supernatants from panel a were further fractionated by densitygradient sedimentation (20–50% sucrose) as described under “Experimental Procedures.” The presence of inhibitor-stabilized soluble Tau species of varying density.*, p 0.1; **, p 0.01; and ***, p 0.001, for comparison of each compound versus the positive aggregation control.

FIGURE 5. Inhibitor-stabilized oligomers resist SDS solubilization. Recom-binant Tau (3 �M) was incubated (16 h, 37 °C) with ODS inducer in the pres-ence and absence of inhibitors (cyanines 1, phenothiazine 10, arylmethine15, or rhodanine 28A; each at 10 �M final concentration), then subjected toSDS-PAGE. a, representative chromatogram visualized by Coomassie Bluestain shows SDS-soluble Tau monomer migrating with apparent molecularmass of 67 kDa. b, quantification of SDS-solubilized Tau monomer remainingafter 16 h incubation (n 3; reported � S.D.), all normalized to DMSO vehiclecontrol. The black bar corresponds to DMSO vehicle control normalized toitself, which was taken to represent 100% SDS solubility. *, p 0.1, and ***,p 0.001, for comparison of each compound versus DMSO vehicle control.The Tau oligomers stabilized by cyanine, phenothiazine, and rhodaninederivatives resisted solubilization in SDS.




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pattern (Fig. 7b). When quantified from replicate analysis (n 3 biological replicates), the null hypothesis was accepted (Fig.7c). These data indicate that the His tag did not mediate inter-action between Tau protein and 1. Therefore, all subsequentexperiments continued to leverage His-tagged Tau constructs

to expedite their purification and analysis. These included aseries of truncations that systematically deleted Tau sequencesfrom the C terminus through the MTBR region (Fig. 7a).Results showed that deletion of all residues C-terminal to thethird (i.e. 2N4R�344) or fourth (i.e. 2N4R�376) MTBR did notaffect the ability of 1 to lower SDS solubility of Tau (Fig. 7, b andc). However, deletion of sequences C-terminal to the secondMTBR (i.e. 2N4R�314) yielded a significant reduction in effi-cacy, whereas deletion before (i.e. 2N4R�252) or after (i.e.2N4R�283) the firstMTBR completely destroyed it (Fig. 7, b andc). Conversely, the MTBR region alone (i.e. 2N4R252–376) sup-ported full efficacy (Fig. 7, b and c). These data revealed thatsequences in the MTBR region, and especially within or adja-cent to the first two imperfect repeats, mediated the loss of SDSsolubility driven by cyanine 1.The MTBR region implicated in inhibitor-Tau interactions

also mediates Tau aggregation propensity (55), suggesting thataggregation intermediates could be direct targets of inhibitoraction. To test this hypothesis, cyanine 1 interactions with2N4R Tau and aggregation-modulating mutants (Fig. 8a) werestudied. First we investigated ODS dependence, because entryinto the aggregation pathway involves conversion of nativelydisordered Tau into aggregation competent conformations,and anionic inducers such as ODS promote this step (reviewedin Ref. 18). Indeed, incubation of 2N4R Tau in the absence ofODS yielded no detectable filaments (data not shown), whereasthe presence of ODS yielded abundant filaments biased towardshorter lengths (Fig. 8b). These data confirmed that detectable2N4R fibrillization was inducer dependent under these condi-tions. In contrast, the presence or absence of ODS and accom-panying fibrillization had no effect on Tau solubility in SDSwhen analyzed by SDS-PAGE (Fig. 8, c and d). However, thepresence of ODS was necessary for Tau SDS solubility to belowered by cyanine 1 (Fig. 8, c and d). These data suggest thatcyanine does not interact efficientlywith natively unfoldedTau,but with conformers populated as a result of interaction withODS micelles.

FIGURE 6. Cyanine-mediated Tau oligomer formation is rapid. Recombi-nant Tau (3 �M) was incubated (37 °C) in the presence (�) or absence (�) of 1(10 �M) without agitation, and aliquots were removed and subjected to SDS-PAGE as a function of time. a, representative gel migration pattern of SDS-soluble Tau monomer visualized by Coomassie Blue stain. The asterisk andarrow marks slowly migrating species appearing at the tops of separating andstacking layers, respectively. b, quantification of SDS-soluble Tau monomerremaining as a function of time, normalized to each DMSO vehicle control(n 3; reported � S.D.). Tau oligomer formation was nearly complete within4 h in the presence of cyanine 1.

FIGURE 7. The Tau microtubule binding repeat region is essential for cyanine-Tau interaction. Recombinant Tau deletion mutants (3 �M) were incubatedwithout agitation (4 h, 37 °C) in the presence (�) or absence (�) of cyanine 1 (10 �M), then subjected to SDS-PAGE. a, map of deletion and missense mutantsused in the analysis, where white bars correspond to deleted regions of Tau protein. The location of MTBRs was mapped as described previously (76). Theconstruct corresponding to the MTBR region alone (2N4R252–376) is referred to as K18a. b, representative gel migration pattern visualized by Coomassie Bluestain. c, quantification of SDS-soluble Tau monomer remaining after 4 h incubation (n 3; reported � S.D.), normalized to each DMSO vehicle control of theconstruct. The black bar corresponds to the 2N4R Tau DMSO vehicle control normalized to itself, which was taken to represent 100% SDS solubility, andtherefore the negative control for inhibitor activity on 2N4R Tau. The blue bar correspond to cyanine-1-mediated depression of SDS-soluble 2N4R Taunormalized to its DMSO vehicle control, and therefore represents the positive control for inhibitor activity on 2N4R tau. ***, p 0.001, for comparison of eachnormalized construct versus 2N4R negative control. #, p 0.05; ###, p 0.001, for comparison of each normalized construct against the 2N4R positive control.Loss of SDS solubility by cyanine required sequences within the MTBR region.




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The second step in the aggregation pathway is reportedlynucleation, which is mediated in part by the 275VQIINK280 (i.e.PHF6*) and 306VQIVYK311 (i.e. PHF6) hexapeptide motifslocated in the MTBR region (56, 57) (Fig. 8a). Missensemutations that introduce Pro residues into these motifs (e.g.2N4RI277P,I308P) have been reported to depress ODS-mediatedfilament formation almost completely (30). Indeed, incubationof 2N4RI277P,I308P with ODS inducer yielded only small aggre-gates devoid of fibrillar morphology (Fig. 8b). Nonetheless,2N4RI277P,I308P supported loss of SDS solubility in the presenceof cyanine 1 (Fig. 8, c and d). These results imply that Tauspecies residing at or beyond the nucleation step are notrequired for 1-mediated effects on Tau solubility.The MTBR region also contains two Cys residues that can

modulate 2N4R Tau aggregation propensity when oxidized(58). Some compounds foster this reaction when incubatedwith Tau under non-reducing conditions (21). To assess thecontribution of Cys residues to cyanine activity, the interactionof 2N4RC291A,C322A, a doublemutant that could not be oxidizedor modified at these positions under any circumstances, wasinvestigated. As reported previously (28), fibrillization of2N4RC291A,C322A resembled wild-type 2N4R Tau, except that asmaller number of longer filaments were produced (Fig. 8b), sug-gesting a lower nucleation rate. Nonetheless, 2N4RC291A,C322Ainteractedwith cyanine1 to yielddepressed levels of SDS-solubleTau (Fig. 8, c and d). This result indicates that cyanine activity isnot mediated solely through Cys oxidation or covalent bindingto these nucleophiles. Overall, these experiments supportcyanine 1 interacting with intermediates stabilized by ODSinducer, but not necessarilywith species at or beyond the nucle-ation stage of the aggregation pathway.

Cyanine Activity Extends to Familial Tauopathy Mutants—In addition to 2N4R Tau, humans express alternatively splicedisoforms that lack products of exons 2, 3, and 10 (Fig. 9a).More-over, certainmissensemutations cause Tau lesion formation infamilial forms of frontotemporal lobar degeneration (59). Asuccessful aggregation inhibitory strategy should extend tothese forms of Tau as well. Therefore, the ability of cyanine 1 todepress SDS solubility of purified Tau isoform 0N3R andtauopathy missense mutants 2N4RR5L, 2N4RG272V, 2N4RP301L,2N4RV337M, and 2N4RR406W was investigated (Fig. 9a). No sta-tistically significant differences were observed between theseproteins and 2N4R Tau with respect to compound-mediateddepression of SDS solubility (Fig. 9, b and c).To test whether depression of SDS solubility correlated with

aggregation inhibitory efficacy, 2N4R, 0N3R, and the tauopathymutants were subjected to aggregation conditions in the pres-ence and absence of 1. Levels of insoluble Tau were then esti-mated after centrifugation. As before, themajority of 2N4RTauwas rendered insoluble when incubated with DMSO vehicle,whereas the presence of 1 significantly depressed recovery ofinsolubleTau (p 0.001, Fig. 9d). In contrast, only aminority offibrillization-incompetent mutant 2N4RI277P,I308P was recov-ered in the pellet fraction in the presence of DMSOvehicle (p0.001 relative to 2N4R), and the presence of 1 did not alter thisdistribution (Fig. 9d). Compared with these boundary exam-ples, 0N3R and the tauopathy missense mutations most closelyresembled 2N4R Tau with respect to recovery of insoluble Tau(Fig. 9d). Although 1 significantly depressed recovery of allinvestigated mutants in insoluble form, the probability ofrejecting the null hypothesis was lowest for 2N4RG272V (p 0.01) and 2N4RP301L (p 0.05), consistent with the especially

FIGURE 8. Cyanine-Tau interaction depends on the presence of inducer. The interaction of cyanine 1 with recombinant 2N4R Tau and missensemutants 2N4RI277P,I308P and 2N4RC291A,C322A was investigated. a, map of missense mutants (Cys 3 Ala and Ile 3 Pro shown in yellow and blue,respectively). b, aggregation behavior of each Tau construct (3 �M) incubated in the presence of ODS (50 �M) without agitation (16 h, 37 °C) observedby transmission electron microscopy (scale bar 500 nm). c, recombinant Tau and missense mutants 2N4RI277P,I308P and 2N4RC291A,C322A (3 �M) wereincubated without agitation (4 h, 37 °C) in the presence or absence of aggregation inducer ODS (50 �M) and cyanine 1, then subjected to SDS-PAGE.Representative migration pattern as visualized by Coomassie Blue stain is shown. d, quantification of SDS-soluble Tau monomer remaining after 16 hincubation (n 3; reported � S.D.), normalized to DMSO vehicle control. The black bar corresponds to the 2N4R Tau DMSO vehicle control normalizedto itself, which was taken to represent 100% SDS solubility, and therefore the negative control for inhibitor activity on 2N4R Tau. The blue barcorresponds to cyanine-1-mediated depression of SDS-soluble 2N4R Tau normalized to the DMSO vehicle control, and therefore represents the positivecontrol for inhibitor activity on 2N4R Tau. ***, p 0.001, for comparison of each condition (gray bars) versus 2N4R negative control. ###, p 0.001, forcomparison of each condition against the 2N4R positive control. Depletion of SDS-soluble Tau species by 1 required the presence of ODS inducer, butnot Cys residues or intact PHF6 and PHF6* sequence motifs.




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high aggregation propensities reported for these species (27).These data indicate that the inhibitory mechanism identifiedherein will likely extend to the multiple Tau species implicatedin neurofibrillary lesion formation.

DISCUSSION

Aggregation Antagonism from the Perspective of Ligand—Many seemingly unrelated Tau aggregation antagonists havebeen disclosed in the academic (7) and patent (5) literatures,some of which show efficacy at high nanomolar/low micromo-lar concentrations. Despite structural diversity, many of thesecompounds share the absorbance characteristics of dyes (i.e.they absorb electromagnetic radiation in the visible spectrum),a property that stems from delocalized -electron distribution(60). Here we found that ligand polarizability, a metric of elec-tron delocalization in the ground state, correlated with inhibi-tory potency within a series of cyanine, phenothiazine, arylme-thine, and rhodanine inhibitors. Although polarizability wasnot the only descriptor of affinity, and by itself was not a pre-dictor of affinity among scaffolds, its modulation may offer aroute for maximizing potency within individual chemotypes.The series investigated here suggest two strategies for doing so.The first is to increase the size of the conjugated -electronnetwork. This approach, which was leveraged in the rhodanineseries, also may contribute to the reported activities of polyene,porphyrin, pthalocyanine, and other large inhibitors (7, 24).

However, polarizability also can be increased through appro-priate positioning of electron donating and withdrawinggroups, as illustrated by the phenothiazine and arylmethineseries. The rank order of potency paralleled the strengths ofconstituent electron donormoieties in both of these series. Thisstrategy has the potential to maximize polarizability whereasminimizing ligand size, and therefore facilitate blood-brain bar-rier penetration (61). For in vivo applications, it will be impor-tant to do so within neutral chemotypes that, unlike the perma-nent cations used herein, support passive diffusion into brain.Aggregation Antagonism from the Perspective of the Tau

Target—Previously, we provided evidence that the ODS-medi-ated Tau aggregation paradigm used to characterize inhibitorsresembles a heterogeneous nucleation pathway (reviewed inRef. 18). In the absence of inhibitors, natively unfolded Tauenters this pathway by interacting with ODS micelles to yieldassembly competent conformations (Fig. 10). Once populated,the rate-limiting step becomes formation of a thermodynamicnucleus on the micelle surface, which then extends by endwiseaddition of free monomers (Fig. 10). Here we found that inhib-itor-mediated disappearance of insoluble Tau filaments wasaccompanied by the appearance of soluble oligomeric com-plexes that resisted denaturation in SDS. Similar Tau oligo-mers were previously reported to form in the presence ofpthalocyanine (24) and also methylene blue (7). More gener-

FIGURE 9. Oligomer-forming efficacy of cyanine inhibitor extends to tauopathy mutants and 3R Tau. Recombinant 2N4R tauopathy mutants 2N4RR5L,2N4RG272V, 2N4RP301L, 2N4RV337M, and 2N4RR406W and Tau isoform 0N3R (3 �M each) were incubated (16 h, 37 °C) with ODS inducer in the presence and absenceof cyanine 1, then subjected to SDS-PAGE. a, map of missense mutants used in the analysis (shown in “orange”) relative to segments derived from alternativelyspliced exons E2, E3, and E10. b, representative SDS-PAGE migration pattern visualized by Coomassie Blue stain. c, quantification of SDS-soluble Tau remainingafter 16 h incubation normalized to DMSO vehicle control (n 3; reported � S.D.). The black bar corresponds to the 2N4R Tau DMSO vehicle control normalizedto itself, which was taken to represent 100% SDS solubility, and therefore the negative control for inhibitor activity on 2N4R Tau. The blue bar corresponds tocyanine-1-mediated depression of SDS-soluble 2N4R Tau normalized to the DMSO vehicle control, and therefore represents the positive control for inhibitoractivity on 2N4R Tau. ***, p 0.001, for comparison of each construct (gray bars) versus the 2N4R negative control. In contrast, no difference (p 0.05) wasdetectable between each construct and the 2N4R positive control, consistent with 1 being capable of depleting SDS-soluble Tau species from all six Tau speciesexamined. d, the products of Tau aggregation reactions (16 h, 37 °C) conducted in the presence of cyanine 1 or DMSO vehicle alone were centrifuged(100,000 � g for 30 min), and the percentage of total Tau protein migrating in the pellet (P) fractions quantified by dot blot analysis using monoclonal antibodyTau5 (n 3; reported � S.D.). *, p 0.1; **, p 0.01; and ***, p 0.001, for comparison of insoluble Tau in the presence versus absence of cyanine 1; ###, p 0.001, for comparison of insoluble Tau in the absence of 1 among Tau constructs. The presence of 1 shifted reaction products out of the pellet fraction for allshown Tau constructs except 2N4RI277P,I308P.




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ally, the phenomenon has been observed with �-synuclein inthe presence of certain polyalcohol, flavonoid, polyene,diazo, porphyrin, and phenoxazine derivatives (25, 62–64).The reciprocal relationship between Tau protein oligomersand filaments suggests they lie off the aggregation pathway(Fig. 10). Sequestration of Tau would be expected to raise theapparent filament critical concentration and to foster end-wise disaggregation of mature filaments, both of which havebeen observed in the presence of cyanine inhibitor (38, 65).Flat, highly polarizable molecules appear to share this solu-ble-oligomer stabilizing activity.The molecular characteristics outlined above are especially

appropriate for interacting with protein surfaces through dis-persion effects (reviewed in Ref. 66). The ODS-mediated Tauaggregation paradigm tested herein yields multiple targets thatcould interact with ligands in this way (Fig. 10). Traditionally,secondary assays for Tau-inhibitor interaction have been usedto identify candidate interacting species residing along thispathway. For example, NMR spectroscopy can detect directinteractions between pthalocyanine and Tau protein (24).More generally, direct interactions between diazo dyes, phe-nothiazines, polyalcohols, and pthalocyanine with monomeric-amyloid (67) and �-synuclein (25, 68, 69) have been reportedas well. However, this approach requires high-micromolar tolow-millimolar concentrations of ligand, and it is not clear thatnatively unfolded protein structures could support such inter-actions at the low protein concentrations used to demonstrateaggregation inhibition. Using thioflavin dye displacement asanother secondary assay, direct interaction between phe-nothiazines, cyanines, and arylmethineswithmatureTau fibrilshas been reported as well (14). Although such binding can behigh affinity, it is difficult to rationalize aggregation inhibitoryactivity in terms of this target alone.In contrast, here we used loss of SDS solubility as the second-

ary assay for Tau-ligand interactions, allowing us to investigatecyanine interactions at near-physiological bulk Tau concentra-tions and reducing conditions. The results showed that nativelyunfoldedmonomer alonewas incapable of supporting oligomerformation. Rather, the absolute requirement for ODS inducersuggested that folded conformations residing along or off theaggregation pathway were the true substrates for inhibitorbinding. Moreover, that intact PHF6 and PHF6* hexapeptide

motifs were not required for interaction with cyanine furthernarrowed candidate binding partners for cyanine inhibitor tothose existing prior to nucleation (Fig. 10). Because we previ-ously reported that polarizability is a descriptor of high affinitybinding to a cross--sheet structure (14), it is tempting to pro-pose that the target has -sheet character. However, �-helicesalso can present appropriate surfaces for binding -delocalizedligands (70), and these too have been proposed to form in con-junction with anionic surfaces (71).Therapeutic Implications—The mechanism of Tau aggrega-

tion inhibitors proposed here has favorable implications fortherapy of tauopathies. First, it can act at physiologically rele-vant bulk Tau concentrations. Indeed, cyanine inhibitors havebeen reported to depress Tau aggregation in ex vivo mousemodels of tauopathy (10, 72). Although oligomer formation hasbeen linked to toxicity in some biologicalmodels (73), the phys-ical characteristics of inhibitor-stabilized oligomers differs (24).Consistent with this hypothesis, treatment of brain slices withcyanine inhibitor did not induce apoptotic responses at the lowconcentrations needed to clear aggregates (10, 72). Second, itsupports interaction with both 3R and 4R isoforms as well asmissense tauopathy mutants, suggesting it could be broadlyapplicable to both AD and frontotemporal lobar degenerationdiseases. Third, it does not rely on interaction with nativelyunfolded Tau monomer, which is an important binding part-ner of microtubules. In fact, �50% of proteins contain longstretches of unfolded structure (reviewed in Ref. 74), whichcould potentially cross-react with inhibitors that target suchregions. Finally, the mechanism predicts that inhibition couldbe useful at early stages of disease before filaments are renderedirreversibly insoluble through cross-linking (75).In summary, we have identified polarizability as a potential

link among structurally diverse Tau aggregation inhibitors. Thecompounds act to rapidly stabilize soluble oligomeric species atthe expense of filamentous aggregates. The proposed mecha-nism suggests design considerations for optimizing inhibitorswith potential therapeutic utility.

Acknowledgments—We thankDhirajMurale andDr. David Church-ill, both of KAIST, Republic of Korea, for providing macrocyclic cya-nine 9, and Dr. Erich Grotewold, the Campus Microscopy and Imag-ing Facility, and Avinash Jaiganesh, The Ohio State University, foraccess to spectroscopy, electron microscopy resources, and assistancewith statistical analysis, respectively.

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Kelsey N. Schafer, Katryna Cisek, Carol J. Huseby, Edward Chang and Jeff KuretStructural Determinants of Tau Aggregation Inhibitor Potency

doi: 10.1074/jbc.M113.503474 originally published online September 26, 20132013, 288:32599-32611.J. Biol. Chem.

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