inhibition of amyloid formation - 2012

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RE V I E W

Inhibition of Amyloid Formation

Torleif Hrd and Christofer Lendel

Department of Molecular Biology, Swedish University of AgriculturalSciences (SLU), Box 590, SE-751 24 Uppsala, Sweden

Received 16 November 2011;received in revised form28 December 2011;accepted 29 December 2011Available online5 January 2012

Edited by S. Radford

Keywords:protein aggregation;amyloidosis;drug discovery;protein engineering;neurodegenerative disease

Amyloid is aggregated protein in the form of insoluble fibrils. Amyloiddeposition in human tissueamyloidosisis associated with a number ofdiseases including all common dementias and type II diabetes. Consider-able progress has been made to understand the mechanisms leading toamyloid formation. It is, however, not yet clear by which mechanismsamyloid and protein aggregates formed on the path to amyloid arecytotoxic. Strategies to prevent protein aggregation and amyloid formationare nevertheless, in many cases, promising and even successful. This reviewcovers research on intervention of amyloidosis and highlights severalexamples of how inhibition of protein aggregation and amyloid formationhas been achieved in practice. For instance, rational design can providedrugs that stabilize a native folded state of a protein, protein engineeringcan provide new binding proteins that sequester monomeric peptides fromaggregation, small molecules and peptides can be designed to blockaggregation or direct it into non-cytotoxic paths, and monoclonal antibodieshave been developed for therapies towards neurodegenerative diseases

based on inhibition of amyloid formation and clearance. 2012 Elsevier Ltd. All rights reserved.

Introduction

Amyloid is insoluble protein in the form of 6- to12-nm-wide and very long unbranched fibrils. Fibrilmorphology can differ, but all amyloid fibrils arehighly ordered witha characteristic cross- X-raydiffraction pattern.1,2 This pattern reflects -sheetsecondary structure arranged so that individual

-strands are perpendicular to the fibril axis.Amyloid has been known for years as proteinaceousmaterial forming insoluble deposits in extracellularspaces of organs and tissues in disease conditionsknown as amyloidosis.3 There are today 27 humanproteins associated with either systemic or localizedamyloidosis and several others give rise to intracel-lular amyloid-like deposits that are associated withdisease.4 Many amyloid diseases are neurodegen-erative and terminal and affect large populations ofelderly, for instance, Alzheimer's disease andParkinson's disease. Others include common disor-ders such as type II diabetes. There is thereforeconsiderable motivation for research on amyloidandon inhibiting cytotoxic effectsof amyloid andproteinaggregation on the path to amyloid formation.

The physical chemistry of amyloid formationappears to be sharedby most, if not all, amyloid-forming proteins.1,5,6 Natively folded proteins donot form amyloidwithout prior unfolding, or at leasttransient local misfolding.7,8 Local misfolding can beself-catalyzed so that a misfolded conformer induces

*Corresponding authors.E-mail addresses:[email protected];[email protected].

Abbreviations used: A, amyloid-protein, amyloid -peptide; ALS, amyotrophic lateral sclerosis; BBB, blood

brain barrier; CNS, central nervous system; FAP, familialamyloid polyneuropathy; HIV, human immunodeficiencyvirus; Hsp, heat-shock protein; IAPP, islet amyloidpolypeptide; MTC, methylthioninium chloride; 248PAP286,peptide with residues 248 to 286 of prostatic acidphosphatase; PrP, prion protein; SAP, serum amyloidprotein; SAR, structureactivity relationship; SOD-1,superoxide dismutase-1; TTR, transthyretin.

doi:10.1016/j.jmb.2011.12.062 J. Mol. Biol. (2012) 421, 441465

Contents lists available at www.sciencedirect.com

Journal of Molecular Biologyj o u r n a l h o m e p a g e : h t t p : / / e e s . e l s e v i e r. c o m . j m b

0022-2836/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
mailto:[email protected]:[email protected]://dx.doi.org/10.1016/j.jmb.2011.12.062http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://dx.doi.org/10.1016/j.jmb.2011.12.062mailto:[email protected]:[email protected]


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similar misfolding in other proteins.8 Unfolded ormisfolded peptides can form soluble aggregates,known as oligomers. Soluble oligomers can form onor off the path to amyloid and they can range in sizeand shape from dimers up to large spherical or fibril-like aggregates (protofibrils), and even annularaggregates.1,9 Different amyloid-forming proteinssometimes show diversity with regard to oligomerformation, but there are indeed common structuralfeatures also of oligomers of different peptides.10,11

The conversion into fibrils with regular cross-secondary structure depends on the formation offibril seeds (nucleation). This is still an enigmaticevent from a mechanistic point of view, but evidencesuggests that it involves concerted conformationalchange of peptides in oligomers or protofibrils.5,12,13

Amyloid fibrils then elongate from seeds by self-templated growth. At this stage, secondary nucle-

ation events also set in. For instance, fibrils undergofragmentation, which increases theirnumberandtheoverall rate of fibril formation.6,14 These molecularevents together account for the characteristic amy-loid formation kinetics in which there is a lag time, asigmoidal growth of fibrils, and a stationary phase.The detailed test-tube kinetics is determined by thepresence of fibril seeds at the outset, the rates ofprimary and secondary nucleation, and the rate offibril elongation.6

The physical mechanism of amyloid formationoffers several possibilities for intervention, as out-lined inFig. 1. One strategy is to either stabilize thenative folded structure by ligand binding or, if there

is no such state, keep the monomeric peptidesequestered by a binding protein from furtheraggregation. Alternatively, aggregate bindingmolecules may be supplied to direct oligomerformation into nonproductive aggregation paths,block fibrils and fibril seeds to prevent elongation(-sheet breaking), or act to disaggregate andremove fibrils and plaques. Externally supplied

agents or endogenous factors may, in this way,keep aggregating peptides away from cytotoxicstates and, in particular, allow for the proteinhomeostasis and immune systems to performbiological clearance of aggregates.

It must be noted at this point that there are fewclear mechanistic links between amyloid depositionand disease pathology. The mechanisms for howprotein aggregation exerts cytotoxic effects arebeginning to be understood,15,16 but whether amy-loid deposition itself in some cases may be protectiveis a subject of discussion.17 Most neurodegenerativeeffects of protein aggregates are now, for instance,thought to be due to smaller soluble oligomericspecies.15,16,18 However, strategies to inhibit proteinaggregation and/or amyloid formation have never-theless shown great promise as potential therapies.

The purpose of this review is to present strategies

for how protein aggregation and amyloid formationcan be inhibited, give an overview of the status ofthe research field, highlight some examples of howinhibition has been achieved in practice, anddescribe some work that has led to further under-standing of aggregation mechanisms. A brief reviewof how amyloid formation is prevented by endog-enous molecular chaperones is included for com-pleteness. However, we do not review mechanismsrelying on surface chemistry, involving, for instance,detergents or lipid membranesand other interfaces.The promise of nanoparticles19 also falls into thiscategory. We provide a referenced table of disease-related proteins discussed in this article (Table 1).

The research field is very large and it cannot becovered in its entirety. (A search of the PubMedarticle database with the key words amyloidandinhibition in November 2011 resulted in morethan 3000 hits.) We therefore aim to cover represen-tative contributions (and contributors), withoutimplying that contributions that have been left outare less significant than those mentioned.

(a) (b) (c) (d) (e)

Native (Partially)

unfolded

Oligomer Amyloid

seed

Amyloid fibril

Refolding Clearance(f) (g)

Fig. 1. Schematic illustration ofstrategies to inhibit the formation ofamyloid fibrils. (a)(e) depict bio-

chemical mechanisms of action thatcould be employed by small orlarge ligands (red) binding to dif-ferent states of amyloid-formingpeptides (blue). (a) Stabilization ofthe native state. (b) Sequestering ofmonomeric peptide. (c) Stabiliza-tion or promotion of off-pathwayoligomers. (d)-Sheet breakers thatterminate fibril elongation. (e) Dis-assembly of amyloid fibrils. Inaddition, refolding (f) and clearance(g) mechanisms (purple) mediated

by chaperones or antibodies can beimportantin vivo.

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Native-State StabilizationProteins or small molecules that bind to the native

folded state of a protein can inhibit its unfolding andaggregation. The thermodynamic basis in an in vitrofoldingunfolding equilibrium is that a molecule,which binds to the native state, will increase thepopulation of this state relative to other (nonnative)states through the law of mass action. However, thepopulation of the folded state can also be underkinetic control. In such a case, it is the folding,unfolding, and misfolding (aggregation) rates thatdetermine the population of the folded state as afunction of time. Proteins or small-molecule binders

can, in this case, protect the folded population bydecreasing the rate of unfolding.

Transthyretin stabilization and the firstanti-amyloid drug

Transthyretin (TTR; previously calledprealbumin)i s a tetrameric protein carrier of thyroid hor-mone.42,45 Familial amyloid polyneuropathy (FAP)is a rare systemic amyloid disease in which muta-tions in TTR lead to endoneurial amyloiddeposits,axonal degeneration, and neuronal loss42 (whereasamyloid formation by TTR in the more common

senile systemic amyloidosis is not associated withmutations43). Nilsson et al. had concluded in 1975that the TTR tetramer exists in equilibrium with themonomer andthat ligand binding acts to stabilizethe tetramer.46 The first low-resolution crystalstructure had been published the year before,47

and Nilssonet al.discuss how tetramer stabilizationcould relate to strengthening of subunitsubunitinteractions by ligand binding to a central cavity inthe tetramer.46

The discovery of a link between FAP genetics andTTR amyloid deposition led to renewed interest inthe biochemistry of TTR. Important contributionswere made by Kellyet al. Biophysical characteriza-

tion confirmed that native tetrameric TTR formsamyloid at moderate acidic conditionsin vitro48 andthat a mutation associated with FAP leads totetramer destabilization and enhanced rate ofamyloid formation.49 Tetramer-to-amyloid conver-sion was shown to occur via a monomeric interme-diate with an altered tertiary structure.50 Petersonetal.determined the structure of TTR in complex withflufenamic acid, and it provided a detailed view ofhow the ligand interacts to tie the dimerdimerinterfaces together.51 The two hormone bindingsites are only partially occupied in serum TTR andtherefore available for small-molecule binding(Ref. 44 and work cited therein). Peterson et al.

proposed that small-molecule inhibitors that stabi-lize the TTR tetramer would be analternative toliver transplantation to treatFAP.51 Rational drugdesign efforts ensued44,5257 and one compound,tafamidis meglumine (Fig. 2), was brought toclinical trials. A pivotal phase II/III trial wascompleted early 2011. If the drug is approved asexpected, it will become the first therapy tosuccessfully treat an amyloid disease by interferingwith amyloid formation.

TTR stabilization by ligand binding is primarilykinetic

Hammarstrm et al. studied the dissociationkinetics and thermodynamic stability of three TTRmutants associated with disease and comparedthese properties to those of wild-type TTR and toclinical features, including disease penetrance andage of onset.58 They found that it is the rate oftetramer dissociation (rather than the stability) thatcorrelates with the severity of disease. In otherwords, tetramer dissociation is rate limiting toamyloid formation. Such a mechanism also explainshow the effect of the V30M TTRmutation, which isthe most prevalent cause of FAP,59 can be rescuedby a T119M mutation on the other allele (Ref.60and

Table 1.Disease-associated proteins and peptides discussed in this article

Protein/peptide Diseases Suggested reading

A Alzheimer's disease 9,15, and22-Synuclein Parkinson's disease, dementia with Lewy bodies, and others 16,18, and2326

-2-Microblobulin Hemodialysis-related amyloidosis 8and27Huntingtin (polyQ) Huntington's disease 28Insulin Injection-localized amyloidosis 29and30IAPP (amylin) Type II diabetes 31Immunoglobulin light chains AL amyloidosis 32Lysozyme Systemic amyloidosis 33and34PrP Spongiform encephalopathies 35and36248PAP286 HIV transmission 37Serum amyloid A AA amyloidosis, familial Mediterranean fever 38SOD-1 ALS 39Tau Alzheimer's disease, frontotemporal dementia, and others 40and41TTR Senile systemic amyloidosis, FAP 4244

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work cited therein). The T119M is not more stablethan wild-type TTR, but this mutant dissociates veryslowly compared to wild type under conditionsfavoring amyloid formation.61 In fact, the presenceof only one T119M TTR subunit in a V30M tetramer

strongly stabilizes the mixed tetramer againstdissociation.60

The effect of TTR amyloid inhibitors is similar tothat of the T119M mutation. They stabilize thetetramer form to which they bind thermodynamical-ly, but the primary effect for amyloid formation, thatis, under conditions at which amyloid is thethermodynamically most stable state, is kinetic. Thisis because binding (and stabilization) of tetramerincreases the free-energy barrier for dissociation andfurther conversion into amyloid. The TTR amyloidinhibitors are therefore referred to as kinetic stabi-lizers of the native state.44,53,61

Stabilization of superoxide dismutase-1

The use of small molecules as pharmacologicalchaperones62,63 to stabilize an aggregation-proneprotein in its native state may be a promisingtherapeutic strategy also for treatment of amyo-trophic lateralsclerosis (ALS), which is a fatal motorneuron disease.39 The most common cause of familialALS is destabilizing mutations in the gene forsuperoxide dismutase-1 (SOD-1). SOD-1 exists as adimer and dimer dissociation precedes unfoldingandamyloid formation. Ray et al. performed in silicoscreening to find drug-like molecules that would

stabilize the dimer interface of SOD-1.64 A series ofchemically unrelated compounds were identified andtested experimentally. These were later improved topotential therapeutics that specifically bind andstabilize SOD-1 in the presence of blood plasma.65

Lysozyme stabilization by a camelid antibody

Binding proteins such as antibodies, single-chainantibody domains, or other proteins selected fromcombinatorial libraries can potentially afford quickroutes to identify native-state stabilizing agents. Ademonstration of the binding-protein strategy isprovided by the inhibition of amyloid formation bya rare variant (D67H)33 of human lysozyme thatcauses hereditary systemic amyloidosis. Dumoulinet al. found that a single-domain fragment of acamelid antibody, which had been selected as a

binder of wild-type lysozyme, inhibits amyloidformation by D67H lysozyme.66

Dumoulinet al.also demonstrated the importanceof restoring global cooperativity of protein foldingin aggregation-prone mutants67 in order to suppressthe population of transient, locally unfolded statesthat occur on the path to amyloid conversion.7,8,66

This is the case for D67H lysozyme stabilization bythe camel antibody, as the antibody does not bind tothe site of the destabilizing mutation, but rathertransmits its effect to that region by stabilizing acooperative network of hydrogen bonding presentin wild-type lysozyme.66

Possible treatment of Parkinson's disease andother neurological diseases

Substantial evidence supports a role of misfoldedforms of-synuclein in theinitiation and progres-sion of Parkinson's disease.16,18 Many studies havetherefore been devoted to the structure and bio-chemistry of -synuclein. However, for the mostpart, these have been based on recombinantlyproduced peptide samples, and they have failed toidentify soluble folded conformations. -Synucleinwas therefore deemed as intrinsically disordered.Bartels et al. recently isolated and characterized

natively folded

-synuclein oligomers with

-helicalsecondary structure.23 Their conclusions are under-pinned by the work of Wang et al.68 The discovery ofnative oligomeric -synuclein opens up for a newtherapeutic strategy in which small molecules orproteins that bind and stabilize the native -synuclein oligomer would be employed to inhibitunfolding and aggregation.

Several other proteins and peptides that areinvolved in neurological disorders are thought tobe intrinsically disordered, for instance, the amy-loid-peptide (A) and tau protein in Alzheimer'sdisease. It is not completely inconceivable that therein fact are natively folded oligomeric forms of these

Fig. 2. Stabilization of the native TTR by a small-molecule ligand. (a) Crystallographic structure of native,tetrameric TTR in complex with 2-(3,5-dimethylphenyl)

benzoxazole (white); Protein Data Bank ID: 2QGE. (b)Chemical structure of tafamidis.



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as well. The therapeutic success with TTR stabiliza-tion and the discovery of the native -synucleinoligomer should motivate the search for such formsso that they too can be targeted by stabilizing agents.It is also potentially possible to stabilize the helicaltransmembrane conformation of A.69

Inhibition of Aggregation by Sequesteringof Monomer

Strong binding to aggregation-prone regions ofamyloidogenic peptides will prohibit self-assembly.Novel binding topologies that completely sequesteraggregation-prone peptide regions may be achievedby protein engineering.

Binding of monomeric A by an engineeredAffibody protein

The selection of binding proteins is now anestablished technology.70 Grnwall et al. usedbiotinylated A40 immobilized on streptavidin-coated beads to select Affibody binders from aphage display library.71 As many as 40 homologousA-binding Affibody molecules were identified(Caroline Grnwall, personal communication; 16sequences were published71).

Hoyer et al. determined the structure of thecomplex between A40and oneof the A-bindingAffibody molecules (ZA3) 72 and studied the

physical chemistry of complex formation.73 Thebasic scaffold, a three-helix bundle, is typicallyconserved in selected binding proteins. However,in this case, ZA3 forms a dimer in which helices 2of the two subunits are linked by a disulfide bond.Helix 1 is destabilized in the selection so that thecores of the two Z-domain scaffolds becomeexposed. This results in the formation of a verylarge tunnel-like binding site in which Abinds as a-hairpin. The hairpin interacts with two -strandsformed by the Affibody subunits to make a four-stranded intermolecular -sheet (Fig. 3).

The thermodynamics and kinetics of A:Affibody

complex formation show that the two peptides firstform an encounter complex in which linked foldingand binding involves a high free-energy barrier withconsequent slow kinetics.73 The equilibrium disso-ciation constant (Kd) of ZA3 for A40 and A42is 17 nM,72 and about 10-fold higher affinity isachieved when the disordered N-terminus of ZA3is removed.74

The apparent propensity for Ato form a hairpinconformation is consistent with a number ofexperimental and theoretical studies. For instance,the hairpin appears when molecular dynamics orMonte Carlo sampling samples conformations.7578

Experimental evidence for the hairpin comes from

nuclear magnetic resonance (NMR) data, and it wassuggested that turnformation of residues 24 to 28nucleates folding.79 Sandberg et al. found that such ahairpin conformation is present also in soluble Aoligomers and protofibrils.80

Inhibition of A aggregation and dissolution ofpreformed aggregates in vitro

A binding by the ZA3 Affibody inhibitsamyloid fibril formation.72,81 The addition of ZA3to Asolutions in which amyloid already is beingformed, for instance, in thelate sigmoidal phase, alsostops further aggregation. Inhibition is achieved on astoichiometric 1:1 basis, which is consistent with thatsequestering of the nonpolar regions at central andC-terminal regions of Aremoves the aggregationpropensity (the 16 N-terminal residues of A areexposed also in the Affibody complex).

The ZA3 Affibody also acts to dissolve pre-

formed aggregates of A

. For instance, largeoligomers (protofibrils) of A42 that travel in thevoid volume in size-exclusion chromatography formfibrils within 10 h (see Ref. 81 for experimentalconditions). However, monomeric A is presenttransiently in this process and the presence ofstoichiometric amounts of Affibody that capturesmonomeric Aacts to dissolve the oligomers. 81 Thekinetics indicates that protofibril dissolution occurson the order of several hours.

Similarly, there appears to be an equilibriumbetween monomeric A and A in fibrils, andZA3 acts to dissolve preformed fibrils. This isbecause Aappears in complex with the ZA3 after

Fig. 3. Structure of the complex between A(orange)and the ZA3 Affibody (backbone of two subunits in blueand cyan, respectively; linking disulfide in yellow).72

Aggregation-prone regions of A are sequestered in atunnel-like nonpolar cavity to form a hydrophobic corethat is common to the Affibody and the Aligand (sidechains displayed as sticks).

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the Affibody has been added to solutions containingpreformed fibrils.81 However, fibril dissolutionkinetics is very slow in physiological buffers indi-cating high kinetic barriers.

Inhibition of Aaggregation and clearance of Ain afruit fly model

Luheshiet al.also examined if the ZA3 Affibodycouldinhibit A aggregation and neurotoxicity invivo.81 The expression of Apeptides in the brainsof transgenic fruit flies (Drosophila melanogaster) istoxic to the flies and generates phenotypes with Aaggregation and other histopathological similaritieswith Alzheimer's disease.82 Flies transgenic withA40 , A42 , o r t h e v e ry aggregation-proneA42E22G (Arctic) mutant83 were made doublytransgenic with ZA3 dimer, monomer, or Z-

domain control. The Affibody dimer was in this

case generated as a head-to-tail construct, toenhance the rate of cysteine disulfide (dimer)formation after translationin vivo.

The median life span of wild-type flies in thisassay is ca 38 days, but it becomes reduced to 9 daysin flies expressing A42E22G. However, the co-expression of the Affibody dimer yields flies with amuch longer median life span (31 days) and thelongevity of flies expressing A42 is completelyrestored from 28 days to wild-type levels. Immuno-histochemistry showed that A aggregates arepresent in the brains of flies that express A butabsent in the brains of those that also express theZA3 dimer. However, the most curious effect is notthat aggregation is inhibited, but that the Affibodyactually acts to clear Afrom the fly.81 The precisemechanism of clearance, that is, if it is intracellularlysosomal or proteasomal or by secretion and

uptake by phagocytic cells, remains unclear.

(a)Inositols

myo- scyllo- epi- chiro-

(c)Rifampicin

(d)Congo red (e)Methylene blue

Fig. 4. Selected small molecules that have been reported to modulate protein aggregation and amyloid formation.

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Small-Molecule Inhibitors of ProteinAggregation

Targeting the misfolding and aggregation of

proteins with small molecules (examples inFig. 4)not only offers great opportunities for drug devel-opment and basic research but also representssignificant challenges. Disease-associated amyloido-genic proteins might seem to be obvious drugtargets. The discovery and design of small moleculeswith the ability to counteract their self-assembly are,however, very different from traditional drugdesign that targets, for instance, the function of anenzyme. Despite the rapid increase of reportsdescribing the inhibition of protein self-assemblyby the means of small molecules, the mechanisms ofaction of many of the compounds are still onlyvaguely understood. Many of the investigatedmolecules appear to act as general anti-amyloidagents with effects reported for several differentamyloidogenic proteins.8486 Although such non-specific behavior is normally not a desirableproperty of drug candidates, the opportunity ofdeveloping broad-spectrum drugs from this type ofmolecules is sometimes mentioned. The significanceof such action remains to be demonstratedand thepresence of functional amyloid in humans 87 maycause complications for this strategy.

Structureactivity relationship of inhibitors ofprotein aggregation

The complexity of the amyloid formation processopens up for a variety of approaches for screeningdepending on what aggregation states or processesare to be targeted. Screening for reduced formationof amyloid fibrils by monitoring the fluorescence ofamyloid binding compounds is frequentlyemployed. Such methodology was, for instance,the basis for an extensive drug discovery effort bythe Mandelkow laboratory to prevent tau proteinaggregation.40 A library of 200,000 compounds wasscreened for inhibition of paired helical filamentformation and dissociation of tau protein aggregates

using a thioflavin S fluorescence-based assay.

88,89

However, relying only on external fluorescentprobes, such as thioflavin T or thioflavin S, hasdrawbacks since interference between the com-pounds under investigation and the reporter mole-cule is often encountered.9093 This was accountedfor in the tau study by a number of controlexperiments, including electron microscopy,intrin-sic tryptophan fluorescence, and filter assays. 88 Thescreen resulted in the identification of 77 com-pounds that could be classified into a few clustersbased on their chemical structure [N-phenylamines,anthraquinones, phenylthiazolylhydrazides, andthioxothiazolidinones (rhodandines)]. From this set

of structures, a detailed investigation of the struc-tureactivity relationship (SAR) of the compoundshas been carried out,40,88,9498 and some generalstructural features, in terms of hydrophobic patchesand hydrogen bonding groups, are emerging.40

Another inhibitor of tau aggregation that hasreceived significant attention is methylene blue (alsoknown as methylthioninium chloride; Fig. 4e).99

Although phase II clinical trials presented in 2008showed remarkable results, the actual mechanism ofaction for this compound is still debated,100102 andit has indeed been reported to also affect Aaggregation.103,104

An interesting example of SAR for inhibitors ofA aggregation is the inositols (Fig. 4a). It wasshown by McLaurinet al.that the inhibiting effect ofthese compounds on A amyloid formation isstrongly dependent on the stereoisomerization and

the distribution of hydroxyl groups on the twosurfaces of the inositol scaffold.105,106 It was foundthat myo-, scyllo-, and epi-inositol but not chiro-inositol promoted the formation of-sheet structureand nontoxic oligomers of A42(but not A40) andthe effect correlated with reduction of toxicity in cellmodels. More recent SAR analysis confirmed theimportance of the presence and arrangement of thehydroxyl groups since scyllo-inositol derivativeswith deletions or substitutions of hydroxyls dis-played reduced ability to counteract fibril formationby A42.

107

The notion that several diseases seem to beassociated with prefibrillar aggregates calls for

more relevant screening criteria than reducedformation of amyloid fibrils. Such effortshave, forinstance, been reported by Neculaet al.108 In parallelwith investigating fibril formation of A42, theyscreened for oligomerformation using a conforma-tion-specific antibody.10 The results revealed thatcertain compounds inhibited the formation ofamyloid fibrils but not oligomers. Other compoundsshowed the opposite behavior, that is, inhibition ofoligomers but not fibrils, and some molecules wereindeed able to inhibit all Aaggregation.

Taken together, the available SAR information isinvaluable for the continuing improvement of

compounds that could be developed into noveltherapeutics. However, it provides limited newunderstanding of the mechanistic features of inhibi-tion of aggregation.

Polyphenols and nonspecific inhibitors

A different screening approach was employed toidentify inhibitors of huntingtin aggregation. Over184,000 compounds were screened and 25 ben-zothiazole derivatives were found to retard aggre-gation of the protein in an automated filterretardation assay.109 Although initial cell-basedstudies looked promising, the lead compound did



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not show good enough results in mouse modelstudies.110 Later on, the same screening methodresulted in the discovery of ()-epigallocatechin-gallate (Fig.4b), a polyphenol present in green tea,asa potent inhibitor of huntingtin aggregation. 111 Thiscompound is indeed fascinating and has later beendemonstrated to interfere with both-synuclein andA aggregation85,112 as well as amyloid-relatedsemen-mediated enhancement of human immuno-deficiency virus (HIV) infection by a fragmentof prostaticacid phosphatase (248PAP286; residues248 to 286)113 and fibrillation of the model protein-casein.114

The class of compounds to which ()-epigalloca-techin-gallate belongs, the polyphenols, are probablythe most frequently investigated molecules when itcomes to inhibition of protein aggregation (forinstance, Refs. 84, 92, 104, and 115-124). Among

many groups, Fink and Uversky et al.have carriedout extensive studies ofthe effect of polyphenols on-synuclein aggregation.125128 From the most recentinvestigations of several flavonoids, it appears thatthe mechanism of action is complex and involvesseveral aspects, including oxidation reactions andcovalent modification.125 There is no direct correla-tion between the binding affinity of thecompoundsand the aggregation inhibition potency125 and thecompounds often stabilize oligomeric states of -synuclein. SAR analysis of available data suggeststhat the arrangement of hydroxyl groups on theflavonoid scaffold is a key issue.126

Another compound that has been reported to

interfere with the aggregation of several differentproteins is rifampicin and its analogues129134 (Fig.4c). This compound is an anti-leprosy drug that wasidentified as an inhibitor of A aggregation afterreports that elderly leprosy patients had reducedamountsof senile plaque compared to age-matchedcontrols.131,133 The anti-amyloid effect of rifampicinwas, in these studies, suggested to be related to aradical scavenger activity. From an extensive inves-tigation of the anti-aggregation effect on-synuclein,the mechanism of action was found to be rathersimilar to that of the flavonoids described above,involving oxidation, potential covalent modifica-

tions, and stabilization of oligomeric species.

129

A fascinating study by Fenget al.draws attentionto a connection between so-called promiscuousinhibitors135,136 and nonspecific inhibitors of proteinaggregation.137 The mechanism of action for thistype of compounds is closely linked to their ability toself-associate and to form supramolecular structures.Indeed, such behavior was recently reported forgeneral aggregation modulators such as Congo red(Fig.4d)andlacmoidwithrespecttotheinhibition ofthe aggregation of-synuclein90 and A.138,139 Theself-stacking ability also seems to correlate very wellwith the inhibitory effect of tetrasulfonatedphtha-locyanines on -synuclein aggregation.140 Hence,

the mechanisms of action for many of the generalanti-amyloidcompounds might resemble those ofdetergents141 and amphipathic molecules142 andthis might explain their broad specificity.

Towards inhibitors of amyloid toxicity andrational design

Kim et al. presented an alternative screeningapproach.143 They used a fusion construct of A42and green fluorescent protein in a cell-based assay,which allowed for effective screening of largelibraries based on the fact that aggregation of A42will also disrupt the native structure of greenfluorescent protein that then loses its fluorescence.This method was demonstrated in a pilot screen of1000 triazine derivatives. An important feature ofthis assay is the ability to identify inhibitors of early

steps of the aggregation process, which is a key issuefor counteracting the formation of oligomers. In theideal case, the compounds should interact with themonomeric peptide in order to prohibit all types ofaggregation. Taking that reasoning one step further,Chenet al.in 2010 presented a screening approachbased on small-molecule microarrays to identifyligands of fluorescently labeled monomeric A40.144Out of the 79 hits from the assay, 15 compoundswere demonstrated to reduce A42-induced cyto-toxicity. Interestingly, when one of these com-pounds was investigated in more detail, it wasfound that it enhanced fibril formation by Aratherthan inhibiting it. Hence, the realization that fibrils

might not be very toxic opens up for fibril formationpromoters and fibril stabilizers as therapeuticagents, and with the increasing knowledge aboutthe aggregation process, it might turn out that smallmolecules are more suitable for targeting the toxicityof certain critical aggregates145,146 rather than thewhole aggregation process.

Moreover, as detailed structural informationabout different types of aggregates is becomingavailable, a more rational approach to design small-molecule ligands will be applicable. A step in thatdirection is the recent results from Landau et al.thatprovide structural models for the interaction be-

tween A

and tau in their amyloid state with smallmolecules by co-crystallization of small peptidefragments with these compounds.147 They foundtwo molecular frameworks of small-molecule bind-ing: one site specific involving charge interactions,which might relate to the observed SAR for inositolsand polyphenol compounds described above, andone more general and nonspecific relying onbinding to tube-like hydrophobic cavities in theamyloid fibrils. Interestingly, binding of orange G tothe A-derived peptide KLVFFA resulted in anantiparallel -sheet orientation in the peptide, whichis also believed to be a structural characteristic of Aoligomers.148,149 This finding might be related to



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that of Necula et al., who found that orange Ginhibited fibril formation but not the formation ofoligomers.108

Advances towards a molecular understanding ofthe interactions between monomeric, intrinsicallydisordered proteins and small-molecule aggrega-tion inhibitors have also been made. Herrera et al.used molecular dynamics simulations to investi-gate the interaction between -synuclein anddopamine,150 amoleculereported to interfere withits aggregation119,151,152 using an NMR-derivedensemble structure. They identified the binding siteand could confirm it experimentally, since mutationofthecriticalE83residuein-synuclein impaired theability of dopamine to inhibit protein aggregation.

Peptide-Based Inhibitors of Amyloid

Growth

The development of peptide-based inhibitors ofamyloid formation has been an intense field ofresearch of which much issummarized in a recentreview by Sciaretta et al.153 Here, we focus onimportant concepts and highlight some more recentdevelopments.

-Sheet breakers: Peptides targeting the centralhydrophobic region of A

The initiation of amyloid formation by Arelies

on interactions involving residues17 to 20 (LVFF;the central hydrophobic region).154 Many efforts todevelop Aaggregation inhibitors have focused onblocking this region and/or finding -sheet breakerpeptides that are unfavorable for further fibrilgrowth.

Tjernberg et al. were first on the scene. 155 Theysynthesized 31 decapeptide fragments on cellulosemembranes and measured binding to radiolabeledA40. They then chose A(1122) for minimization.The shortest peptide (35) that still showed signifi-cant binding was KLVFF, corresponding to A(1620), and residues corresponding to wild-type posi-

tions 16, 17, and 20 could not be replaced with analanine without loss of A40 binding. An elegantexperiment supported their design strategy: A40with triple K16A, L17A, and F20A mutations doesnot easily form amyloid fibrils. Screening of combi-natorial libraries ofD-amino acid pentapeptides ledto the identification of several inhibitors andTjernberg et al. suggested that protease-resistant D-amino acid peptides may be used as inhibitingagents towards amyloid formation in vivo.156

Around the same time, Soto et al.used a differentdesign strategy to develop -sheet breaker pep-tides that inhibit amyloid formation.157 They fo-cused directly on the central hydrophobic region

containing the LVFFA motif of A and designedpeptides with similar sequences and hydropho-bicity. Proline residues hadpreviously been notedto reduce -sheet propensity158 in Aand prolineswere therefore inserted in the inhibitor peptides,while charged residues were added at the peptideends to increase solubility. The iA1 peptide indeedinhibits amyloid formation and in fact also promotesdisaggregation of preformed A40and A42fibrils.Interestingly, a protease-resistant D-amino acidhomolog of iA1 also inhibits amyloidogenesis.One of the other peptides (iA5; sequence LPFFD)protects neuroblastoma cell cultures from Atoxicity and reduces cerebral deposition of A invivo.159 A variant of this peptide was later shown toprevent A-induced spatial memory impairments ina rat model.160

Following the early developments by Tjernberg

et al.and Soto et al., peptides targeting the centralhydrophobic region of A became leadsequencesfor optimization of A amyloid inhibitors.161170 Forinstance, it was shown that the KLVFF recognitionelement could be combined with disruptive ele-ments such as cationic lysines in KLVFFKKKK andsuch compounds canbe effective also at substoichio-metric concentrations.162 It has also been shown thatmultivalencyof the KLVFF motif increases inhibitorefficiency.164 It is furthermore possible to enhancethe activity of peptide inhibitors of Aaggregation,such as LVFFA, through modification of the aminotermini by organic reagents, such as cholic acid. 171

The strategy to block aggregation by partially

complementary peptides has also been applied tothe prion protein (PrP), -synuclein, islet amyloidpolypeptide (IAPP), and insulin.172175

-Sheet breaker peptideswith modified backbones

The -sheet breakers described above weredeveloped as complementary peptides or peptideswith fibril disrupting side chains. A drawback withsuch inhibitors has, in some cases, been that theythemselves form amyloid fibrils. An alternativedesign strategy is to block -sheet extension infibrils by inhibiting intermolecular backbone hydro-

gen bond formation. A very attractive backbonemodification is N-methylation, because it alsoachieves steric hindrance for amyloid formationand because N-methyl amino acid residues areconstrained to adopt -strand conformations. Anillustrating property of N-methylated peptides asinhibitors of amyloid formation is that they are verysoluble in water.153 They are also potentiallymembrane penetrating and can diffuse rapidlythrough phospholipidbilayers.176

The N-methylation strategy was first employed byHughes et al., who demonstrated that five of sixsingle N-methyl derivatives ofA(2535) inhibitedfibril formation and cytotoxicity.177 The concept was



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extended by incorporating N-methylation at everysecond peptide to create a true -sheet cap with anN-methyl face that is incompatible with furtheramyloid fibril elongation of A.178 A very effectiveinhibitor of Aaggregation was identified by usingthe KLVFF peptide described above as a startingpoint to create a library of peptides with differentlengths, chiralities, N- and C-terminal modifica-tions, side-chain identities, and backbone N-meth-ylation status.179 N-methylated peptides have alsobeen successfully developed as inhibitors of -synuclein180,181 and IAPP182,183 amyloid formation.

Structure-based peptide inhibitor design

The design of inhibitors of amyloid formationdescribed above has been based on intelligentinferences about the effect of various peptide

segments on amyloid formation, such as sterichindrance of-sheet formation and blocking of-strand hydrogen bonding. They have, despiteconsiderable progress, been challenging due to lackof detailed structural information and the designedinhibitors do, to some extent, lack specificity.

The most detailed information on amyloid fibrilstructure to date is the many fibril-like crystalstructures of short peptides determined by theEisenberg laboratory.184186 These have allowed fora range of fundamental insights regarding amyloidformation, for instance, that the amyloid core islikely to be a steric zipper with very tight side-chain interdigitation of opposing faces of the -

sheets. They also allow for structure-based de-velopments of novel specific amyloid inhibitors, asdemonstrated by the designof nonnatural peptideinhibitors by Sieverset al.187 Essentially, the conceptis to use existing structures to design and optimize

peptides that are compatible with cross- confor-mations on one side and incompatible with thepropagation of such structures on the other side.That is, peptides are designed to cap the growingamyloid in much the same way as other peptideinhibitors mentioned in the previous section. How-ever, there are two major differences to previousapproaches. First, as mentioned, inhibitors aredesigned using high-resolution structures of modelamyloid fibrils, and second, the design process relieson the optimization possibilities afforded by theRosetta software for prediction of protein structureand proteinprotein interactions.188,189

Two nonnatural amino acid peptide inhibitorshave so far been described: an all-D-amino acidinhibitor of fibril formation by the tau protein and anonnatural L-amino acid inhibitor of aggregation of248PAP 286. The target for the tau aggregation

inhibitor was the fibril-like crystal structure of thehexapeptide VQIVYK, corresponding to residues306311 of tau. This peptide was chosen because it isessential to fibril formation and because it formsfibrils on its own. The model of the aggregating PAPfragment was the structure of the GGVLVNhexapeptide that had been predicted190 to formsteric-zipper aggregates. The underlying assump-tion in both cases was that the steric-zipperstructures observed in crystals represent the coresof amyloid fibrils.

The most effective of the inhibitors of tau aggre-gation (an all-D-TLKIVW peptide) was designed tobind with maintained main-chain hydrogen bonding

at the top of the VQIYVK steric-zipper structure, butwith D-Leu2 inhibiting additional VQIYVK bindingto both the inhibitor itself and to the opposing strandin the steric zipper (Fig. 5). Thioflavin T fluorescenceassays showed thatD-TLKIVW significantly delayed

Fig. 5. Structure-based rational design of peptide inhibitors of amyloid formation. The designed D-TLKIVW peptide(red and blue) is modeled into the crystal structure of VQIVYK steric zipper (magenta and gray). Protein Data Bank ID:2ON9. The steric clash caused by D-Leu2 (yellow star) prohibits further growth of the fibril. (a) View perpendicular tofibril axis. (b) View with fibril axis perpendicular to the figure plane. Reprinted with permission from MacmillanPublishers Ltd.:Nature(Ref.187, Supplementary Fig. 3), copyright 2011.



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(or inhibited) amyloid formation by tau proteinfragments. Electron microscopy was used to showthat it preferentially binds to ends of fibrils, asintended, and NMR experiments indicate an equi-librium dissociation constant of ca 2M. There is nohigh-resolution structure of the inhibitorfibril com-plex yet, but all experiments and in particularaggregation assays with variants of the inhibitorindicate that it functions as intended.

The design strategy for inhibiting 248PAP 286

aggregation into amyloid was to include the use ofnonnatural amino acids in an all-L hexapeptide.Hence, the contact area with the target peptide wasincreased by the use of alanine and tyrosine/prolinederivatives (chAla and hydroxyTic, respectively).The designed peptide (called WW61) is a veryefficient inhibitor of amyloid formation by248PAP286; a two-molar excess blocks seeded aggre-

gation for more than 2 days. The WW61 inhibitorwas subsequently shown to also inhibit 248PAP286-mediated HIV infection in a cell culture assay.

In summary,Sievers et al. nicely demonstrate a newstrategy for amyloid inhibitor development in whichspecificity of the inhibition is the key achievement.

Peptide inhibitor selection from combinatoriallibraries

The possibility of selecting inhibitors of amyloidformation from libraries of random peptides wasfirst explored by Nagai et al., who selected trypto-phan-rich peptides that bind to aggregation-prone

polyglutamines related to, for instance, Hunting-ton's disease.191 This group was also among the firsttoexplorethefruitfly(Drosophila)asan in vivobenchtest for amyloid inhibitors, and they demonstratedthat the most efficient peptide inhibits polygluta-

mine-induced damage and premature death in aDrosophila model.192 Several groups have subse-quently employed combinatorial libraries to selectshort peptides that block amyloid formation and, insome cases, stabilize non-amyloid aggregates.193197

(Phage display and related techniques are alsocommonly used to select binding proteins, asdiscussed in other sections of this article.)

Phage display selection of D-amino acid inhibitorsof Aaggregation

Peptides based on L-amino acids are, as men-tioned, vulnerable to protease digestion and shortlivedin vivo. However, it is not possible to select D-amino acid peptides from biological libraries such asphage libraries. One may circumvent this problemusing mirror image phage display in which L-form

peptidebinders are selected using a D-form targetprotein.198 The corresponding D-amino acid pep-tides are then, by symmetry binding, the naturallyoccurring (L-form) target with the same affinity. Thistechnique was employed by Willbold et al. todirectly select D-amino acid peptide inhibitors ofAaggregation.199201 Selection from a large libraryof 12-mer peptides at very low (2 nM) concentra-tions led to the identification of binders rich inarginine and histidine residues. The dominantpeptide (called D3), with the curious sequenceRPRTRLHTHRNR, inhibits A aggregation andcytotoxicity in vitro. I t can also cross a modelbloodbrain barrier (BBB)202 and indeed acts to

reduce Aplaque loadina transgenic mouse modelof Alzheimer's disease.199 It will be very interestingto learn what the mechanism of biological activity ofD3 is, as well as the role of the many cationicresidues in this mechanism.

Fig. 6. At least three differentmechanisms of action have beensuggested for A-targeted immu-notherapy. (a) The peripheral sinkhypothesis states that binding ofantibodies to monomeric Ain theperiphery (blood) alters the equilib-rium and transport of Aover theBBB. (b) Antibodies that cross theBBB into the CNS might interferedirectly with the aggregation pro-cess of A. (c) Antibodies thatrecognize amyloid fibrils can triggerclearance of plaque via Fc-mediatedphagocytosis.

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Antibody-Mediated Inhibition andImmunotherapy

Immunotherapy is one of the most promising

routes to new anti-amyloid treatments, and much ofthe current knowledge about the formation ofamyloid and the potential strategies to interferewith these processes in vitro and in vivooriginatesfrom antibody-based research.203

A immunotherapy

The development of immunotherapy targeting ofA has been particularly successful, and a number ofantibodies are currently in the late stages of clinicaltrials.20 Many of these have inhibitory effects on theAaggregation process in vitro.149,204207 The posi-

tive effects of anti-A immunotherapy in vivo is,however, believed to be the result of slightlydifferent mechanisms of action, as described belowand illustrated inFig. 6.

Using active immunization of A42in a transgenicmouse model overexpressing human amyloid pre-cursor protein, Schenk et al. demonstrated in 1999that the generated anti-Aantibodies were able toprevent Aplaque formation in young animals aswell as to reduce A-related neuropathologicalchanges in old animals.208 Other studies confirmedcognitive improvements due to active vaccination inmouse models.209,210 Following investigations ofanti-A polyclonal and monoclonal antibodies

showed that similar results could be achieved withpassive immunization.211 The presented data sug-gested that the antibodies were able to cross the BBBand act within the central nervous system (CNS) andthat amyloid clearance was achieved via Fc-mediat-ed phagocytosis by microglia.211,212 In addition, theresults suggested that only antibodies against the N-terminal region of A recognized and promotedclearance of A plaque.212 This finding indeedcorrelates very well with previous in vitro obser-vations204 and the fact that the N-terminus is notinvolved in the core of A amyloid fibrils andtherefore should be accessible to antibody binding.21

DeMattos et al. suggested an alternative mecha-nism of action in 2001 based on their findings that amonoclonal antibody recognizing the central regionof Avery efficiently sequestered Ain plasma.213

By creating aperipheral sinkand thereby shiftingthe equilibrium concentrations of Ain plasma andcerebrospinal fluid, efflux of A from the CNS couldbe promoted and amyloid deposition could bereduced. Indeed, the antibody appeared to lowerbrain A deposits in the employed mouse modelalthough cerebrospinal fluid levels of soluble Aincreased. This study did not find any evidence ofbinding of the antibody to amyloid plaque, whichcan be attributed to the fact that the A epitope

recognized by this antibody might be hidden withinthe fibrils. Since then, both the central clearance andthe peripheral sink mechanisms have been exten-sively investigated, questioned, and adjusted (forinstance, Refs.214-216). However, the exact mech-anism of action of A-targeting immunotherapyremains elusive.20

Targeting protein aggregates

With emerging insights about the critical roles ofprefibrillar aggregates in Alzheimer's disease andother disorders, the attention has been directedtowards antibodies that recognize conformationalepitopes on oligomeric species.217,218 Significantadvances in this area were made by Glabe and co-workers. They were first to demonstrate that it ispossible toachieve selective binding to soluble A

oligomers.10

Interestingly, the polyclonal A11 anti-body that they obtained also recognize soluble(prefibrillar) oligomers of other amyloid-formingproteins and it reduces their cytotoxicity, suggestingthat a common conformational epitope might beinvolved in cytotoxic mechanisms. Further workresulted in the isolation of antibodies with selectivebinding to fibrils andfibrillar oligomers219 and toannular protofibrils,220 and they too recognizesimilar species formed by proteins with differentprimary structure. In addition, the generation ofmonoclonal antibodies against prefibrillar oligomersrevealed a range of immunologically distinct sub-populations of such oligomers.221

A similar finding was made by O'Nuallain andWetzel when generating antibodies to Aamyloidfibrils.222 These antibodies recognize amyloid fibrilsfrom a variety of different proteins (2-microglu-bulin, IAPP, TTR, huntingtin, lysozyme, and animmunoglobulin VL domain) but not the corre-sponding native forms or non-amyloid proteinaggregates. Interestingly, it was later found thatantibodies with general amyloid-recognitionprop-erties can be isolated from human sera.206 Thesewere shown to bind specifically to amyloid fibrils ofat least five different proteins (A, serum amyloidA protein, immunoglobulin light chain, TTR, and

IAPP), and they effectively reduce the formation ofamyloid fibrils of all these proteins.A molecular basis for such broad selectivity was

recently suggested from the investigation of anantibody selected by phage display to bind Aamyloid fibrils.149 The binding of this antibody wasfound to rely on highly ordered clusters of negativecharges at the amyloid surface, and besides bindingto a variety of amyloid fibrils, it also interacts withother types of negatively charged polymers, such asDNA or heparin.223,224

However, not all amyloid/oligomer bindingantibodies display broad recognition patterns. Amonoclonal antibody raised against protofibrils of



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Awith the Arctic mutation (E22G) was shown tobe selective for protofibrillar Aand not bind otherA species or amyloid of other proteins, demon-strating the existence of unique epitopes of Aaggregates that are distinct from more generalamyloid epitopes.80,225 This antibody was alsoshown to inhibit Aaggregation andto effectivelyclear soluble Aaggregatesin vivo.207

An interesting finding regarding the oligomerselectivity of anti-A antibodies was recentlyreported by Lindhagen-Persson et al.205 Theyshowed that a monoclonal IgM antibody (with 10antigen binding regions) raised against monomericA40 preferentially binds oligomeric A. The au-thors attribute this to avidity effects. The antibodywas also shown to inhibit fibril growth and reduceA-induced cytotoxicity. Furthermore, the authorsdemonstrated that oligomer selectivity seems to be a

common property among IgM autoantibodiesagainst A isolated from humans. Indeed, severalstudies have found that autoantibodies recognizingamyloidogenic proteins, including A, PrP, and -synuclein, normally exist in humans.205,226232 Al-though their origin and function are not yetunderstood, such antibodies have shown promisefor therapy and diagnostics.206,226,233,234

Other amyloidogenic proteins as antibody targets

A offers rather unique opportunities amongthe neurodegenerative misfolding disorders sinceit is mainly found in extracellular plaque. This is

a feature that it has in common with systemic andlocalized amyloidosis and it has, for instance,been shown that monoclonal antibodies canresolve immunoglobulin light-chain amyloid in amouse model.235 The majority of the neurodegen-erative disorders, however, are associated withintracellular inclusions. Nevertheless, accumulat-ing data show that immunotherapy could still beeffective, which indicates that the target proteinsare exposed at certain points during the progres-sion of the diseases. Intracellular expression ofantibodies (intrabodies) in combination with suit-able delivery techniques might also be a feasible

approach.236

Substantial efforts have been put into the devel-opment of immunotherapy approaches with the PrPas target.35 Peretz et al. showed that recombinantFab fragments interfered with prion replication andcleared existing PrPSc in cell culture.237 The sug-gested mechanisms of action involved binding of theantibodies to certain epitopes on PrPC, which wouldinterfere with potential interactions with PrPSc andthereby inhibit prion propagation. A number ofstudies have since then confirmed the positiveeffects of PrPC- and/or PrPSc-binding antibodies incell models (for instance, Refs.237-242) and preven-tion of prion pathology in animal models.243,244

Despite the fact thatin vitrostudies have revealedmoderate or no effect on -synuclein aggregationby antibodiesbinding to the C-terminal part of theprotein,245,246 active and passive immunotherapybased on such approacheshave shown promisingresults in animal studies.247,248 Similar to theN-terminus of A,theC-terminalpartof-synucleinis believed to be outside the fibrillar core,24,249 andthe studies indeed indicate that activation ofphagocytic degradation is critical for the in vivomechanism of action of these antibodies. Antibodybinders to the central, so-called NACregion, of-synuclein250 and to oligomeric species251 have alsobeen generated and shown to counteract aggrega-tion of the protein in vitro. Immunotherapeuticapproaches are also explored for huntingtin252255and tau.232,256258

Finally, it is notable that the targets of antibodies

do not have to be the amyloidogenic protein itself.Bodin et al. recently demonstrated that antibodiesraised against serum amyloid protein (SAP), whichassociates with amyloid deposits of several differentproteins, efficiently clear amyloid via phagocytosisin a mouse model.259 A key aspect of this work is theparallel use of a small molecule that binds andpromotes the clearance of circulating SAP.260,261

This clearance enables the antibody to reach SAP inthe amyloid deposits.

Nature's Own Way: Molecular Chaperonesand the Proteostasis Network

The physical chemistry of protein folding,unfolding, and aggregation (kinetics and thermo-dynamics) is in vivoacting in concert with a rangeof other mechanisms in the protein homeostasis(proteostasis) network to control the pool of foldedprotein in various cellular and extracellular com-partments.262 In a recent review, Powers et al.propose a conceptual framework in which theproteostasis network interprets and alters proteinfolding energetics to keep the protein pool within aproteostas is boundary (essentially out o f trouble).263 The proteostasis boundary is deter-

mined by native-state stability and folding/misfold-ing rates, as well as by factors controlling localconcentration, such as protein production, traffick-ing, and degradation, and molecular chaperones aspart of the protein quality control system.264,265 Theinterconnection between proteostasis and proteinfolding energetics implies that the proteostasisnetwork can also be targeted by chemical andbiological agents to prevent formation and accumu-lation of amyloid.262,266 While this promising field ofresearch is outside the scope of the present review,we will describe in this sectionsome of the molecularchaperones that have been studiedin their roles asinhibitors of amyloid formation.267



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The extracellular clusterin, 2-macroglobulin,and haptoglobin chaperones

Also known by many other names, includingapolipoprotein J, clusterin is a stress-induced mo-lecular chaperone that is expressed in all humantissues, including the brain (see Ref. 268 for anextensive review). Clusterin is a secreted protein,but a number of intracellular, and even nuclear,forms have been reported. There are several iso-forms, but secreted clusterin consists of two pep-tides (a 34-kDa -subunit and a 47- kDa -subunit)that are linked by five disulfides. The matureheterodimer is N-glycosylated at six sites andsialylated.

Clusterin colocalizes with extracellular amyloiddeposits in at least 11 disorders. 269 Experiments invitro show that clusterin binds to prefibrillar

aggregates to prevent further aggregation andamyloid formation.268,269 The structural mecha-nisms of clusterin's interaction with client proteinsare yet to be characterized in detail. However, thepromiscuous binding activities have been attributedto three regions of natively disordered or moltenglobule-like structures, containing putative amphi-pathic -helices, which can act as adaptable bindingsites.270 Wyatt et al. studied interactions betweenclusterin and three proteins subjected to heatstress.271 They found that clusterin complexes arevery large (N40,000 kDa). The complexes havedifferent stoichiometries, but the mass ratio remainsconstant at 1:2 (clusterin:client). An analysis of

secondary-structure content in these complexessuggested that clusterin might interact with avariety of protein unfolding intermediates.

Clusterin is expressed in the brain in response toinjury and the protein was already noted in earlystudies to be overexpressed in neurodegenerativedisease, including Alzheimer's disease (Refs. 268and272 and work cited therein). However, a cleargenetic link between clusterin polymorphism andAlzheimer's was not identified until recently, whennew genetic evidence suggested that late-onsetforms are also associated with defective clearanceof the A peptide.273 In fact, the clusterin gene

(CLU) is listed as number three among geneticdeterminants associated with Alzheimer's disease(after APOE and BIN1). A role of clusterin in Aclearance fits well with thein vitrobiochemistry, andthe molecular biology of clearance is beginning tobecome understood. There is considerable evidencefrom experiments in vivo that clusterin and apoli-poprotein E have similar functions in the clearanceof A aggregates from the brain22,268 and binding ofA42to clusterin almostdoubled its rate of transportover the BBB in mouse.22

Two other secreted and abundant glycoproteins,2-macroglobulin and haptoglobin (also known fortheir protease trapping and hemoglobin bindingactivities, respectively), act on a range of aggregatingproteins by mechanisms that are similar to that ofclusterin. It has been suggested that all three proteinsact to maintain protein aggregates in a soluble stateuntil they can be removed from the extracellularspace by receptor-mediated endocytosis.274

The Hsp70 chaperone

Hsp70 (heat-shock protein 70) is one of the centralmolecular chaperones in cytosolic proteostasis,where it acts together with the Hsp40 co-chaperoneand nucleotide exchange factorsto promote proteinfolding and prevent aggregation.265,275 Briefly, ATP-bound Hsp70 binds to hydrophobic segments of

unfolded, partially folded, misfolded, and/or pre-aggregated peptide substrates. Hydrolysis of boundATP into ADP then converts Hsp70 to a conforma-tion in which the peptide binding affinity increases.Both initial binding and ATP hydrolysis are cata-lyzed by Hsp40. Nucleotide exchange factors cata-lyze the release of ADP and rebinding of ATP byHsp70 recycles it to the low-affinity state in which anunfolded or partially folded substrate is released.Fast-folding proteins will bury hydrophobic seg-ments upon release and slow-folding molecules willrebind to Hsp70 (rather than form aggregates) to beallowed another chance to fold. 265 Conformationalchanges and unfolding of misfolded species can also

be facilitated in the Hsp70-bound state.275The precise kinetic balance between the alterna-tives of Hsp70 binding or aggregation can bechallenged under stress conditions, but it can insuch cases be restored by expression of morechaperone via the stress-response pathway.265

Hsp70/40 chaperone overexpression can, in thisway, rescue cells from thetoxicitycausedonthepathto amyloid formation262,276 and inhibit amyloidformation in vitro.277,278 Dedmonet al.showed thatHsp70 alone (without other factors) also inhibitsamyloid fibril formation by -synuclein in vitro atsubstoichiometric concentrations.279 The mecha-

nism is one in which Hsp70 binds to prefibrillaraggregates to prevent maturation of these into fibrilsand possibly also allow them to dissociate intosmaller species. Hsp104, which otherwise also isATP dependent, displays similar activity on Aaggregation.280

Hsp70 may indeed be active to prevent -synuclein aggregation and/or toxicity in the extra-cellular space. The protein does not contain asecretion signal and it is therefore not secreted bygeneral mechanisms. However, Hsp70 is secreted byother mechanisms from a large number of cell typesin response to stress, to provide, for instance,neuronal protection and to play a role in inflamma-www.alzgene.org

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tory and immune responses.281284 Danzer et al.recently studied oligomers of -synuclein in theextracellular space of a cellular model of neurons anddemonstrated that they can be trans-synapticallytransmitted from cell to cell.285 They found thatextracellular Hsp70 acts to modulate this activity andrescue trans-synaptic toxicity. These findings motivatefurther studies on the role of extracellular Hsp70 in thebiology of neurological disease and suggest that -synuclein modulation by Hsp70 in this wayrepresentsa potential new target for therapeutic intervention.285

Other molecular chaperones

ATP-dependent molecular chaperones, such asHsp70, and Hsp90 and chaperonins (Hsp60) pro-mote protein folding by ATP hydrolysis. Non-ATP-dependent intracellular chaperones, such as Hsp27

andB-crystallin of the small Hsp family, also act tobuffer aggregation and amyloid formation, perhapsin a way similar to that of the non-ATP-dependentactivities of Hsp70 and Hsp104 described above. Forinstance, B-crystallin inhibits amyloid formationby both -synuclein286 and A42,287 and the abilityof Hsp27 to inhibit fibril formation by A42has beenknown for some time. 288

Conclusions and Perspective

The critical roles of protein aggregates in agrowing number of devastating diseases have

during the last decades promoted a massivescientific interest for the amyloid phenomenon. Aquestion of significant importance within this fieldof research is how the self-association of proteins canbe modulated by extrinsic factors, such as smallmolecules, peptides, or antibodies. In this review,we have highlighted a range of examples toillustrate the possible mechanisms of action of suchmolecules. The described work also illustrates howcareful investigation of amyloid inhibition cangenerate new insights into the fundamental chem-istry of protein aggregation as well as of thepathologies of the associated disorders.

Even more important is the potential to developnew therapies. Today, a few anti-amyloid strategieshave made it to the late stages of clinical trials, andwe may soon have the first approved drug targetingamyloid formation. This is a historic advance thatcarries great hope for the future. At the same time,many of the basic mechanisms of action of anti-amyloid compounds remain to be studied in detailto elucidate the most efficient approaches tocounteract protein aggregates therapeutically. Isthe inhibition of the aggregation process the rightway to go, and which types of aggregates are thenthe most critical to avoid? Or will it be more effectiveto target the processes by which the aggregates

cause toxicity, either by triggering clearance of toxicspecies or by interfering with the biochemicalprocesses leading to cellular damage? Characteriz-ing the cytotoxicity pathways is indeed one of themain challenges in the field, and an improvedunderstanding of these will be of critical importancefor optimizing the therapeutic action of inhibitors ofamyloid formation.

Acknowledgement

This work was supported by grants from theSwedish Research Council (VR).

References1. Chiti, F. & Dobson, C. M. (2006). Protein misfolding,

functional amyloid, and human disease. Annu. Rev.Biochem. 75, 333366.

2. Greenwald, J. & Riek, R. (2010). Biology of amyloid:structure, function and regulation. Structure, 18,12441260.

3. Sipe, J. D. & Cohen, A. S. (2000). Review: history ofthe amyloid fibril.J. Struct. Biol. 130, 8898.

4. Sipe, J. D., Benson, M. D., Buxbaum, J. N., Ikeda, S. I.,Merlini, G., Saraiva, M. J. M. & Westermark, P.(2010). Amyloid fibril protein nomenclature: 2010recommendations from the nomenclature committeeof the International Society of Amyloidosis. Amyloid,

17, 101104.5. Caughey, B. & Lansbury, P. T. (2003). Protofibrils,pores, fibrils, and neurodegeneration: separating theresponsible protein aggregates from the innocent

bystanders.Annu. Rev. Neurosci. 26, 267298.6. Knowles, T. P., Waudby, C. A., Devlin, G. L., Cohen,

S. I., Aguzzi, A., Vendruscolo, M. et al. (2009). Ananalytical solution to the kinetics of breakablefilament assembly.Science, 326, 15331537.

7. Dhulesia,A.,Cremades,N.,Kumita,J.R.,Hsu,S.T.D.,Mossuto, M. F., Dumoulin, M. et al. (2010). Localcooperativity in an amyloidogenic state of humanlysozyme observed at atomic resolution. J. Am. Chem.Soc. 132, 1558015588.

8. Eichner, T., Kalverda, A. P., Thompson, G. S.,

Homans, S. W. & Radford, S. E. (2011). Conforma-tional conversion during amyloid formation atatomic resolution.Mol. Cell, 41, 161172.

9. Roychaudhuri, R., Yang, M., Hoshi, M. M. & Teplow,D. B. (2009). Amyloid -protein assembly andAlzheimer disease.J. Biol. Chem. 284 , 47494753.

10. Kayed, R., Head, E., Thompson, J. L., McIntire, T. M.,Milton, S. C., Cotman, C. W. & Glabe, C. G. (2003).Common structure of soluble amyloid oligomersimplies common mechanism of pathogenesis. Sci-ence, 300, 486489.

11. Bolognesi, B., Kumita, J. R., Barros, T. P., Esbjrner, E.K., Luheshi, L. M., Crowther, D. C.et al.(2010). ANS

binding reveals common features of cytotoxic amy-loid species.ACS Chem. Biol. 5, 735740.



16/25

12. Cheon, M., Chang, I., Mohanty, S., Luheshi, L. M.,Dobson, C. M., Vendruscolo, M. & Favrin, G. (2007).Structural reorganisation and potential toxicity ofoligomeric species formed during the assembly ofamyloid fibrils.PLoS Comput. Biol. 3, e173.

13. Lee, J., Culyba, E. K., Powers, E. T. & Kelly, J. W.(2011). Amyloid-forms fibrils by nucleated confor-mational conversion of oligomers.Nat. Chem. Biol. 7,602609.

14. Masel, J., Jansen, V. A. & Nowak, M. A. (1999).Quantifying the kinetic parameters of prion replica-tion.Biophys. Chem. 77, 139152.

15. Querfurth, H. W. & LaFerla, F. M. (2010). Alzheimer'sdisease.N. Engl. J. Med. 362, 329344.

16. Winklhofer, K. F., Tatzelt, J. & Haass, C. (2008). Thetwo faces of protein misfolding: gain- and loss-of-function in neurodegenerative diseases. EMBO J. 27,336349.

17. Treusch, S., Cyr, D. M. & Lindquist, S. L. (2009).Amyloid deposits: protection against toxic protein

species?Cell Cycle, 8, 16681674.18. Winner, B., Jappelli, R., Maji, S. K., Desplats, P. A.,Boyer, L., Aigner, S.et al.(2011). In vivo demonstra-tion that -synuclein oligomers are toxic. Proc. Natl

Acad. Sci. USA, 108, 41944199.19. Kanwar, J. R., Sun, X., Punjd, V., Sriramojua, B.,

Mohane, R. R., Zhouf, S. F. et al. (2012). Nanoparticlesin the treatment and diagnosis of neurologicaldisorders: untamed dragon with fire power to heal.Nanomedicine, 8, 399414.

20. Pul, R., Dodel, R. & Stangel, M. (2011). Antibody-based therapy in Alzheimer's disease. Expert Opin.Biol. Ther. 11, 343357.

21. Petkova, A. T., Ishii, Y., Balbach, J. J., Antzutkin,O. N., Leapman, R. D., Delaglio, F. & Tycko, R.

(2002). A structural model for Alzheimer's -amyloid fibrils based on experimental constraintsfrom solid state NMR. Proc. Natl Acad. Sci. USA, 99,1674216747.

22. Bell, R. D., Sagare, A. P., Friedman, A. E., Bedi, G. S.,Holtzman, D. M., Deane, R. & Zlokovic, B. V. (2007).Transport pathways for clearance of human Alzhei-mer's amyloid -peptide and apolipoproteins E and Jin the mouse central nervous system. J. Cereb. BloodFlow Metab. 27, 909918.

23. Bartels, T., Choi, J. G. & Selkoe, D. J. (2011). -Synuclein occurs physiologically as a helically foldedtetramer that resists aggregation. Nature, 477,107110.

24. Heise, H., Hoyer, W., Becker, S., Andronesi, O. C.,

Riedel, D. & Baldus, M. (2005). Molecular-levelsecondary structure, polymorphism, and dynamicsof full-length -synuclein fibrils studied by solid-state NMR. Proc. Natl Acad. Sci. USA, 102,1587115876.

25. Uversky, V. N. (2007). Neuropathology, biochemis-try, and biophysics of -synuclein aggregation. J.Neurochem. 103, 1737.

26. Lee, V. M. & Trojanowski, J. Q. (2006). Mechanismsof Parkinson's disease linked to pathological-synuclein: new targets for drug discovery.Neuron,52, 3338.

27. Heegard, N. H. (2009). 2-microglobulin: fromphysiology to amyloidosis.Amyloid, 16, 151173.

28. Ross, C. A. & Tabrizi, S. J. (2011). Huntington'sdisease: from molecular pathogenesis to clinicaltreatment.Lancet Neurol. 10, 8398.

29. Dische, F. E., Wernstedt, C., Westermark, G. T.,Westermark, P., Pepys, M. B., Rennie, J. A. et al.

(1988). Insulin as an amyloid-fibril protein at sites ofrepeated insulin injections in a diabetic patient.Diabetologia, 31, 158161.

30. Groenning, M., Frokjaer, S. & Vestergaard, B. (2009).Formation mechanism of insulin fibrils and structur-al aspects of the insulin fibrillation process. Curr.Protein Pept. Sci. 10, 509528.

31. Westermark, P. A., Andersson, A. & Westermark,G. T. (2011). Islet amyloid polypeptide, islet amyloid,and diabetes mellitus.Physiol. Rev. 91, 795826.

32. Buxbaum, J. & Gallo, G. (1999). Nonamyloidoticmonoclonal immunoglobulin deposition disease.Light-chain, heavy-chain, and light- and heavy-chain deposition diseases. Hematol. Oncol. Clin.North Am. 13, 12351248.

33. Pepys, M. B., Hawkins, P. N., Booth, D. R., Vigushin,D. M., Tennent, G. A., Soutar, A. K. et al. (1993).Human lysozyme gene mutations cause hereditarysystemic amyloidosis.Nature, 362, 553557.

34. Booth, D. R., Sunde, M., Bellotti, V., Robinson, C. V.,Hutchinson, W. L., Fraser, P. E. et al. (1997).Instability, unfolding and aggregation of humanlysozyme variants underlying amyloid fibrillogen-esis.Nature, 385, 787793.

35. Aguzzi, A. & Sigurdson, C. J. (2004). Antiprionimmunotherapy: to suppress or to stimulate? Nat.Rev., Immunol. 4, 725736.

36. Aguzzi, A. & Calella, A. M. (2009). Prions: proteinaggregation and infectious diseases.Physiol. Rev. 89,11051152.

37. Mnch, J., Rcker, E., Stndker, L., Adermann, K.,Goffinet, C., Schindler, M. et al. (2007). Semen-derived amyloid fibrils drastically enhance HIVinfection.Cell, 131, 10591071.

38. Westermark, G. T. & Westermark, P. (2009). Serumamyloid A and protein AA: molecular mechanismsof a transmissible amyloidosis. FEBS Lett. 583,26852690.

39. Ferraiuolo, L., Kirby, J., Grierson, A. J., Sendtner, M.& Shaw, P. J. (2011). Molecular pathways of motorneuron injury in amyotrophic lateral sclerosis. Nat.Rev. Neurol. 7, 616630.

40. Bulic,B.,Pickhardt,M.,Schmidt,B.,Mandelkow,E.M.,Waldmann, H. & Mandelkow, E. (2009). Developmentof tau aggregation inhibitors for Alzheimer's disease.

Angew. Chem., Int. Ed. Engl. 48, 17401752.41. Morris, M., Maeda, S., Vossel, K. & Mucke, L. (2011).The many faces of tau. Neuron, 70, 410426.

42. Hou, X., Aguilar, M. I. & Small, D. H. (2007).Transthyretin and familial amyloidotic polyneuro-pathy. Recent progress in understanding the molec-ular mechanism of neurodegeneration. FEBS J. 274,16371650.

43. Westermark, P., Sletten, K., Johansson, B. &Cornwell, G, G., III (1990). Fibril in senile systemicamyloidosis is derived from normal transthyretin.Proc. Natl Acad. Sci. USA, 87, 28432845.

44. Johnson, S. M., Wiseman, R. L., Sekijima, Y., Green,N. S., Adamski-Werner, S. L. & Kelly, J. W. (2005).



17/25

Native state kinetic stabilization as a strategy toameliorate protein misfolding diseases: a focus onthe transthyretin amyloidoses. Acc. Chem. Res. 38,911921.

45. Rask, L., Peterson, P. A. & Nilsson, S. F. (1971). The

subunit structure of human thyroxine-binding pre-albumin.J. Biol. Chem. 19, 60876097.46. Nilsson, S. F., Rask, L. & Peterson, P. A. (1975).

Studies on thyroid hormone-binding proteins. II.Binding of thyroid hormones, retinol-binding pro-tein, and fluorescent probes to prealbumin andeffects of thyroxine on prealbumin subunit selfassociation.J. Biol. Chem. 250, 85548563.

47. Blake, C. C. F., Geisow, M. J., Swan, I. D. A., Rerat,C. & Rerat, B. (1974). Structure of human plasmaprealbumin at 2.5 resolution. A preliminary reporton the polypeptide chain conformation, quaternarystructure and thyroxine binding. J. Mol. Biol. 88,112.

48. Coln, W. & Kelly, J. W. (1992). Partial denaturation

of transthyretin is sufficient for amyloid fibrilformation in vitro. Biochemistry, 31, 86548660.49. McCutchen, S. L., Coln, W. & Kelly, J. W. (1993).

Transthyretin mutation Leu-55-Pro significantly al-ters tetramer stability and increases amyloidogeni-city.Biochemistry, 16, 1211912127.

50. Lai, Z., Coln, W. & Kelly, J. W. (1996). The acid-mediated denaturation pathway of transthyretinyields a conformational intermediate that can self-assemble into amyloid.Biochemistry, 35, 64706482.

51. Peterson, S. A., Klabunde, T., Lashuel, H. A., Purkey,H., Sacchettini, J. C. & Kelly, J. W. (1998). Inhibitingtransthyretin conformational changes that lead toamyloid fibril formation. Proc. Natl Acad. Sci. USA,95, 1295612960.

52. Johnson, S. M., Connelly, S., Wilson, I. A. & Kelly,J. W. (2009). Toward optimization of the second arylsubstructure common to transthyretin amyloidogen-esis inhibitors using biochemical and structuralstudies.J. Med. Chem. 52, 11151125.

53. Connelly, S., Choi, S., Johnson, S. M., Kelly, J. W. &Wilson, I. A. (2010). Structure-based design of kineticstabilizers that ameliorate the transthyretin amyloid-oses.Curr. Opin. Struct. Biol. 20, 5462.

54. Almeida, M. R., Gales, L., Damas, A. M., Cardoso, I.& Saraiva, M. J. (2005). Small transthyretin (TTR)ligands as possible therapeutic agents in TTRamyloidoses.Curr. Drug Targets CNS Neurol. Disord.4, 587596.

55. Gales, L., Macedo-Ribeiro, S., Arsequell, G., Valencia,

G., Saraiva, M. J. & Damas, A. M. (2005). Humantransthyretin in complex with iododiflunisal: struc-tural features associated with a potent amyloidinhibitor.Biochem. J. 388, 615621.

56. Palaninathan, S. K., Mohamedmohaideen, N. N.,Orlandini, E., Ortore, G., Nencetti, S., Lapucci, A.et al. (2009). Novel transthyretin amyloid fibrilformation inhibitors: synthesis, biological evaluation,and X-ray structural analysis. PLoS ONE, 4, e6290.

57. Kolstoe, S. E., Mangione, P. P., Bellotti, V., Taylor,G. W., Tennent, G. A., Deroo, S. et al. (2010).Trapping of palindromic ligands within nativetransthyretin prevents amyloid formation. Proc.Natl Acad. Sci. USA, 107, 2048320488.

58. Hammarstrm, P., Jiang, X., Hurshman, A. R.,Powers, E. T. & Kelly, J. W. (2002). Sequence-dependent denaturation energetics: a major determi-nant in amyloid disease diversity. Proc. Natl Acad.Sci. USA, 99, 1642716432.

59. Saraiva, M. J. (1996). Molecular genetics of familialamyloidotic polyneuropathy. J. Peripher. Nerv. Syst.1, 179188.

60. Hammarstrm, P., Schneider, F. & Kelly, J. W. (2001).Trans-suppression of misfolding in an amyloiddisease.Science, 293, 24592462.

61. Hammarstrm, P., Wiseman, R. L., Powers, E. T. &Kelly, J. W. (2003). Prevention of transthyretinamyloid disease by changing protein misfoldingenergetics.Science, 299, 713716.

62. Cohen, F. E. & Kelly, J. W. (2003). Therapeuticapproaches to protein-misfolding diseases. Nature,426, 905909.

63. Morello, J. P., Salahpour, A., Laperrire, A., Bernier,V., Arthus, M. F., Lonergan, M. et al. (2000).

Pharmacological chaperones rescue cell-surface ex-pression and function of misfolded V2 vasopressinreceptor mutants.J. Clin. Invest. 105, 887895.

64. Ray, S. S., Nowak, R. J., Brown, R. H. & Lansbury, P.T. (2005). Small-molecule-mediated stabilization offamilial amyotrophic lateral sclerosis-linked super-oxide dismutase mutants against unfolding andaggregation. Proc. Natl Acad. Sci. USA, 102,36393644.

65. Nowak, R. J., Cuny, G. D., Choi, S., Lansbury, P. T. &Ray, S. S. (2010). Improving binding specificity ofpharmacological chaperones that target mutantsuperoxide dismutase-1 linked to familial amyo-trophic lateral sclerosis using computationalmethods.J. Med. Chem. 53, 27092718.

66. Dumoulin, M.,Last, A. M.,Desmyter, A.,Decanniere,K., Canet, D., Larsson, G. et al. (2003). A camelidantibody fragment inhibits the formation of amyloidfibrils by human lysozyme.Nature, 424, 783788.

67. Dumoulin, M., Canet, D., Last, A. M., Pardon, E.,Archer, D. B., Muyldermans, S.et al.(2005). Reducedglobal cooperativity is a common feature underlyingthe amyloidogenicity of pathogenic lysozyme muta-tions.J. Mol. Biol. 346, 773788.

68. Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A.,Nguyen, L. T. T., Liao, J. et al.(2011). A soluble -synuclein construct forms a dynamic tetramer. Proc.Natl Acad. Sci. USA, 108, 1779717802.

69. Nerelius, C., Sandegren, A., Sargsyan, H., Raunak,R., Leijonmarck, H., Chatterjee, U. et al. (2009).-helix targeting reduces amyloid-peptide toxicity.Proc. Natl Acad. Sci. USA, 106, 91919196.

70. Grnwall, C. & Sthl, S. (2009). Engineered affinityproteinsgeneration and applications. J. Biotechnol.140, 254269.

71. Grnwall, C., Jonsson,A.,Lindstrm, S.,Gunneriusson,E., Sthl, S. & Herne, N. (2007). Selection andcharacterization of Affibody ligands binding to Alzhei-mer amyloidpeptides.J. Biotechnol. 128, 162183.

72. Hoyer, W., Grnwall, C., Jonsson, A., Sthl, S. &Hrd, T. (2008). Stabilization of a -hairpin inmonomeric Alzheimer's amyloid- peptide inhibitsamyloid formation. Proc. Natl Acad. Sci. USA, 105,50995104.



18/25

73. Hoyer,W. & Hrd, T. (2008). Interaction of Alzheimer'sA peptide with an engineered binding proteinthermodynamics and kinetics of coupled folding-

binding.J. Mol. Biol. 378, 398411.74. Lindgren, J., Wahlstrm, A., Danielsson, J., Markova,

N., Ekblad, C., Grslund, A.et al.(2010). N-terminalengineering of amyloid--binding affibody mole-cules yields improved chemical synthesis and higher

binding affinity. Protein Sci. 19, 23192329.75. Lam, A. R., Teplow, D. B., Stanely, H. E. & Urbanc, B.

(2008). Effects of the Arctic (E22G) mutation onamyloid -protein folding: discrete molecular dy-namics study.J. Am. Chem. Soc. 130, 1741317422.

76. Mitternacht, S., Staneva, I., Hrd, T. & Irbck, A.(2010). Comparing the folding free-energy landscapeof A42 variants with different aggregation proper-ties.Proteins, 78, 26002608.

77. Sgourakis, N. G., Yan, Y., McCallum, S. A., Wang, C.& Garcia, A. E. (2007). The Alzheimer's peptidesA40 and 42 adopt distinct conformations in water: a

combined MD/NMR study. J. Mol. Biol. 368 ,14481457.78. Yang, M. & Teplow, D. B. (2008). Amyloid-protein

monomer folding: free-energy surfaces reveal allo-form-specific differences.J. Mol. Biol. 384, 450464.

79. Lazo, N. D., Grant, M. A., Condron, M. C., Rigby, A.C. & Teplow, D. B. (2005). On the nucleation ofamyloid -protein monomer folding.Protein Sci. 14,15811596.

80. Sandberg, A., Luheshi, L. M., Sllvander, S., Pereirade Barros, T., Macao, B., Knowles, T. P. et al.(2010).Stabilization of neurotoxic Alzheimer amyloid-oligomers by protein engineering. Proc. Natl Acad.Sci. USA, 107, 1559515600.

81. Luheshi, L. M., Hoyer, W., Barros, T. P. d., Hrd,

I. v. D., Brorsson, A. C., Macao, B. et al. (2010).Sequestration of the A peptide prevents toxicityand promotes degradation in vivo. PLoS Biol. 8,e1000334.

82. Crowther, D. C., Kinghorn, K. J., Miranda, E., Page,R., Curry, J. A., Duthie, F. A. I. et al. (2005).Intraneuronal A, non-amyloid aggregates andneurodegeneration and in a Drosophila model ofAlzheimer's disease.Neuroscience, 132, 123135.

83. Nilsberth, C., Westlind-Danielsson, A., Eckman, C.B., Condron, M. M., Axelman, K., Forsell, C. et al.(2001). The Arctic APP mutation (E693G) causesAlzheimer's disease by enhanced A protofibrilformation.Nat. Neurosci. 4, 887893.

84. Masuda, M., Suzuki, N., Taniguchi, S., Oikawa, T.,

Nonaka, T., Iwatsubo, T.et al.(2006). Small moleculeinhibitors of-synuclein filament assembly.Biochem-istry, 45, 60856094.

85. Ehrnhoefer, D. E., Bieschke, J., Boeddrich, A., Herbst,M., Masino, L., Lurz, R. et al.(2008). EGCG redirectsamyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat. Struct. Mol. Biol. 15,558566.

86. Frid, P., Anisimov, S. V. & Popovic, N. (2007). Congored and protein aggregation in neurodegenerativediseases.Brain Res. Rev. 53, 135160.

87. Maji, S. K., Perrin, M. H., Sawaya, M. R., Jessberger,S., Vadodaria, K., Rissman, R. A. et al. (2009).Functional amyloids as natural storage of peptide

hormones in pituitary secretory granules. Science,325, 328332.

88. Pickhardt, M., Gazova, Z., von Bergen, M.,Khlistunova, I., Wang, Y., Hascher, A. et al. (2005).Anthraquinones inhibit tau aggregation and dissolve

Alzheimer's paired helical filaments in vitro and incells.J. Biol. Chem. 280, 36283635.89. Pickhardt, M., von Bergen, M., Gazova, Z., Hascher,

A., Biernat, J., Mandelkow, E. M. & Mandelkow, E.(2005). Screening for inhibitors of tau polymeriza-tion.Curr. Alzheimer Res. 2, 219226.

90. Lendel, C., Bertoncini, C. W., Cremades, N.,Waudby, C. A., Vendruscolo, M., Dobson, C. M.et al. (2009). On the mechanism of nonspecificinhibitors of protein aggregation: dissecting theinteractions of -synuclein with Congo red andLacmoid.Biochemistry, 48, 83228334.

91. Buell, A. K., Dobson, C. M., Knowles, T. P. &Welland, M. E. (2010). Interactions between amyloi-dophilic dyes and their relevance t

inhibition of amyloid formation - 2012

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