Plaque busters: strategies to inhibit amyloid formation in Alzheimer's disease

ALZHEIMER’S disease (AD) is the most common cause of late-life de-mentia and represents the fourth leading cause of death in the developedworld. AD is a progressive degenerative disorder of insidious onset andis characterized by memory loss, confusion and a variety of cognitivedisabilities. Clearly, this disease is a major social and health-care prob-lem for which there is no cure or effective treatment. AD can be dividedinto a familial (autosomal dominant) form (FAD), which usually has anearly onset (before 60 years), and a sporadic late-onset form.

The major neuropathological changes in the brains of AD patients areneuronal death, particularly in regions related to memory and cognition,and the presence of abnormal intra- and extracellular proteinaceous fil-aments1. Intracellularly, bundles of paired helical filaments, composedlargely of phosphorylated tau protein and referred to as neurofibrillarytangles, accumulate in large numbers in dying neurons1. Extracellularly,insoluble aggregates of proteinaceous debris, termed ‘amyloid’, appear

in the form of senile or neuritic plaques and cerebrovascular amyloiddeposits. The frequency and distribution of neurofibrillary tangles andneuritic plaques appear to correlate well with the extent of cognitive im-pairment, synaptic loss and neurotransmitter depletion, which are alsocharacteristic of AD (Ref.1). This review focuses on the potential role ofamyloid in the pathogenesis of AD and discusses current strategies tominimize the negative effect of amyloid in the brain.

Amyloid deposition as a central event in the pathogenesis of ADAmyloid deposits mainly comprise aggregates of a 39–42 residue pep-tide called amyloid-b peptide (Ab), which is encoded on chromosome21 as a larger protein, termed amyloid precursor protein (APP)2,3. The evi-dence that Ab aggregates play an important role in the early pathogene-sis of AD comes primarily from studies of individuals affected by thefamilial form of the disease or by Down syndrome. Almost all patientswith Down syndrome (trisomy 21) develop AD neuropathology at anearly age4. In a survey of brains from Down syndrome patients betweenthe ages of 25 and 60 years, diffuse amyloid deposits were observed inyounger subjects in the absence of any other cerebral abnormality4, in-dicating that the formation of amyloid deposits is perhaps the first neu-ropathological alteration. Furthermore, the fact that mutations in theAPPgene are associated with familial AD is in itself a strong indicationof the importance of amyloid in the pathogenesis of the disease5.

In addition to APP, three other genes have so far been linked withAD (Refs 2,6): apolipoprotein E (apoE, chromosome 19), presenilin 1(PS1, chromosome 14) and presenilin 2 (PS2, chromosome 1).Mutations or polymorphisms in these genes induce an increase in theproduction or amyloidogenicity of Ab (Refs 2,6–8) and therefore ap-pear to be implicated in the disease through the formation of amyloid.

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Rev iewsMOLECULAR MEDICINE TODAY, AUGUST 1999 (VOL. 5)

1357-4310/99/$ - see front matter © 1999 Elsevier Science. All rights reserved. PII: S1357-4310(99)01508-7

Claudio Soto PhDAssociate Professor, University of Chile

Dept of Biology, Faculty of SciencesLas Palmeras 3425, Santiago, Chile

and Senior ScientistSerono Pharmaceutical Research Institute14 Chemin des Aulx,1228 Plan les Ouates

Geneva, SwitzerlandTel: 141 22 706 9851

Fax: 141 22 794 6965 E-mail: [email protected]

Plaque busters: strategies toinhibit amyloid formation inAlzheimer’s disease

Claudio Soto

AlzheimerÕs disease is a devastating degenerative disorder of the brain for which there is no cure oreffective treatment. Although the etiology of AlzheimerÕs disease is not fully understood, recent researchsuggests that deposition of cerebral amyloid plaques is central to the disease process. Therefore, anattractive therapeutic strategy for AlzheimerÕs disease is to prevent, reduce or reverse amyloid depositionin the brain. Several small chemical compounds, synthetic peptides and natural proteins have beendescribed that inhibit amyloid formation or amyloid neurotoxicity in vitro. The effect of these and othercompounds now needs to be tested in vivo and the ability of amyloid inhibitors to halt the progression ofAlzheimerÕs disease in humans needs to be evaluated.

Moreover, transgenic mice that express high levels of human mutantAPPprogressively develop many of the pathological hallmarks of AD,including cerebral amyloid deposits,neuritic dystrophy , gliosis,neuronal loss and behavioral alterations9. Thus, it seems that amyloidis an early event in AD and perhaps causative of the other brain abnor-malities that typify the disease (Fig. 1). However, it is important to notethat the available evidence can be alternatively interpreted: it is possi-ble that the toxic state of Ab is not the final amyloid plaque but ratheran altered Ab intermediate in solution, which is a precursor of theamyloid fibrils (Fig. 1). In this scenario, the formation of amyloid de-posits could be regarded as a protective event. The most direct evalu-ation of the actual contribution of amyloid to Alzheimer’s dementiawill come from experiments that analyze disease progression after in-hibiting or reversing amyloid deposition.

APP is a transmembrane glycoprotein, and the Ab domain startsclose to the membrane on the extracellular side and ends within themembrane2. The APP gene contains at least 19 exons, and .10 iso-forms of APP are generated by alternative splicing2,3,5. As the Ab se-quence arises from portions of exons 16 and 17, it cannot be generatedby alternative splicing but requires proteolytic cleavage at both the N-and the C-terminus. Because APP has to be cleaved within the mem-brane to release Ab, it was speculated that abnormal APP processing ormembrane damage was the first pathological alteration in AD (Ref. 3).However, it is now clear that low levels of a peptide identical to theone deposited in AD amyloid are constitutively produced by most celltypes10, and Ab is a normal constituent of human biological fluids11,12.

These findings suggest that abnormal APPprocessing is not required for amyloid forma-tion and that Ab fibrillogenesis might be theearliest pathological event in the disease (Fig.1). Regardless of whether soluble Ab (sAb) isthe precursor of the fibrillar Ab, it is certainthat the same sequence can adopt two differentstates with distinct structural and biologicalproperties. The key question in AD amyloido-genesis is why Ab is soluble under certainconditions but aggregates under others.

Preventing amyloid deposition: atherapeutic target for ADPrevention of the negative influence of amy-loid seems an attractive therapeutic approachfor the disease. Other therapeutic strategies indevelopment (such as cholinesterase in-hibitors, muscarinic, glutamatergic or sero-tonergic agonists, anti-inflammatory drugs,estrogen-replacement therapy and anti-oxi-dant drugs) are directed towards palliating existing cognitive symptoms or retarding thedisease course. The advantage of an amyloid-related therapy for AD is that it is targeted atcorrecting or preventing the pathology be-lieved to cause the disease; therefore, it mightchange the course of the disease or (if used atthe appropriate time) even prevent its onset.Preventing amyloid deposition will also allowevaluation of the actual role of amyloid inAlzheimer’s dementia. There are several pos-sible targets to lower the cerebral amyloid

burden in AD (Fig. 2). These are summarized below.

Reduction of APP gene expression It is conceivable that the expression of APPcould be prevented, or atleast lowered, by using modern techniques of gene therapy, such as an-tisense oligonucleotides or genetically engineered ribozymes.Although promising, the effective application of gene therapy tech-niques is still under development. Furthermore, until the biologicalfunction of APP is established, it remains possible that lowering APPproduction might produce undesirable side effects. Indeed, homozy-gous App-deficient mice develop various abnormalities, including decreased body weight, reduced locomotor activity, spontaneousseizures, impaired behavioral performance and premature death13. Inaddition, the App null animals show reactive astrogliosisand a de-crease in synaptophysin, indicating that APP might play an importantrole in synaptic function in central nervous system (CNS) neurons13.

Alteration of APP processingAb is generated from APP by two proteolytic cleavage events that in-volve the activity of at least two enzymes, named b- and g-secretase2.Inhibition of these enzymes or activation of non-amyloidogenic APPprocessing will probably reduce the brain concentration of sAb andhence slow the formation of amyloid. Extensive effort has been spentin the last 10 years to identify and inhibit the enzymes involved in APPprocessing. Although several enzymes have been proposed to play arole in Ab production, so far there is no consensus on the identity of

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Figure 1. A working hypothesis for the progression of AlzheimerÕs disease (AD). Although the final cause ofdementia is the extensive neuronal loss in brain areas related with memory, the available evidence suggeststhat conformational changes in soluble amyloid-b peptide (sAb) that result in amyloid plaque formationmight be the triggering event in the disease. The critical role of Ab misfolding links AD to several other etio-logically diverse disorders where protein conformational changes play a central role in the disease process.Abbreviations: APP, amyloid precursor protein; ApoE4, apolipoprotein E4.

APP cleavage sAbProduction of more�amyloidogenic sAb

Increased sAbproduction

Oxidation

Metal ions

Trisomy 21APP�

mutations

APP�mutations

Presenilins�mutations

Presenilins�mutations

Local factors (pH,�free radicals, etc.)

ApoE4 and other�pathological chaperons

Conformationally�altered Ab

Amyloid fibrils

Vascular amyloid

Neuritic plaques

Neurofibrillary�tangles

Neuronal�damage

DementiaMolecular Medicine Today

b- or g-secretase activity. It is likely that several proteases are impli-cated in APP processing and, therefore, inhibition of their activity willbe difficult and most likely result in serious side effects.

Inhibition of Ab amyloidfibril formationThe available evidence indicates that Ab aggregation and cerebral amy-loid deposition are probably the first pathological processes in AD. Thisfact and the abundant knowledge accumulated in the molecular mecha-nism of amyloid formation14,15make the inhibition of Ab amyloidogen-esis an attractive therapeutic target for AD. However, the lack of knowl-edge on which Ab species (amyloid plaque or a b-sheet-rich precursorintermediate) is toxic to the brain makes this target difficult, unless inhi-bition of the very early steps in amyloid formation is achieved.

Acceleration of the clearance of amyloid plaques or sAbVery little is known about the physiological mechanisms of amyloid re-moval and sAb catabolism in the brain. This is in part owing to thelack of appropriate models to study in vivo clearance mechanisms. Ithas been proposed that post-translational chemical modifications ofAb are responsible for inefficiency of peptide removal in AD brains, re-sulting in increased amyloid deposition8. In fact, several chemicalmodifications have been detected in Ab deposited in amyloid, includ-

ing oxidation, deamination, racemization anda substantial amount of pyroglutamylation atthe N-terminal region8. On the other hand, it isbelieved that amyloid deposits are very resis-tant to removal, owing to their high insolubil-ity, the attachment of a number of amyloid-binding proteins and the presence of variousdegenerating cell types, including dystrophicneurites, astrocytes and microglial cells8. Thelack of basic knowledge of the clearancemechanism makes this process a difficult tar-get for the production of therapeutic com-pounds.

Prevention of amyloid neurotoxicity Despite considerable progress in the under-standing of the diverse aspects of amyloido-genesis in AD, the mechanism by which amy-loid is involved in neurodegeneration is stillunclear. As Ab fibrils are toxic to neuronalcells in culture, it has been proposed thatamyloid could be directly implicated in AD-associated neurodegeneration. Neurotoxicityis dependent on the formation of amyloid-likedeposits; amorphous Ab aggregates are nottoxic16. This observation is consistent withneuropathological studies of AD brain, whichhave shown that neurodegenerative changesoccur around compact amyloid plaques, butnot around diffuse deposits composed of non-fibrillar A b aggregates17. However, the mole-cular mechanism of in vitro Ab neurotoxicityis still not clear. Several different mechanismshave been proposed to mediate amyloid toxi-city17, including: (1) an increase in intracellu-lar Ca21; (2) impairment of mitochondrialredox activity resulting in the increase of free

radicals; (3) formation of ion channels; and (4) activation of signaltransduction by interaction with specific cellular receptors17. It remainsto be determined whether Ab neurotoxicity is mediated by a singlecentral mechanism or by several mechanistically distinct pathways.

Molecular and structural basis of Ab fibrillogenesisSynthetic peptides homologous to Ab have been used extensively tostudy fibril formation in vitro14,15. Peptides containing the sequence1–40 or 1–42 and their shorter derivatives form amyloid-like fibrils invitro, which are morphologically, tinctorially, immunologically, spec-troscopically and ultrastructurally similar to fibrils extracted from ADamyloid plaques.

A fruitful approach in the study of the relationship between the structure and fibrillogenicity of Ab has been to alter the sequence of thepeptide and to analyze the effect on amyloid formation. Replacing hydrophobic for hydrophilic residues in the internal Ab hydrophobic re-gion (amino acids 17–21) impairs fibril formation18, suggesting that Abassembly is driven partially by hydrophobic interactions. This idea is con-sistent with the higher amyloidogenicity of Ab peptides that have two orthree additional hydrophobic amino acids at the C-terminal end ofAb1–40 (Ref. 19). On the other hand, several reports indicate that the con-formation adopted by Ab peptides is crucial in amyloid formation20–22.

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Figure 2. Target sites for an amyloid-related therapy for AlzheimerÕs disease (AD). The cerebral amyloid bur-den could be decreased by targeting different but related processes. The putative negative effect of amyloidin the brain might be minimized by: (a) reducing amyloid precursor protein (APP) expression; (b) altering APPprocessing; (c) inhibiting amyloid fibril formation; (d) accelerating the clearance of amyloid/soluble amyloid-b peptide (sAb); or (e) preventing amyloid neurotoxicity. Each of these targets has advantages and disad-vantages that have to be considered when designing a drug directed to reduce the effect of amyloid in AD.

APP gene

Chromosome 21Cell membrane

Neuronal death�and dementia

Amyloid fibrils

sAb circulating�in human fluids

b Altering APP processing

c Inhibiting amyloid formation

d Accelerating amyloid or sAb clearance

e Preventing amyloid neurotoxicity

APPAb

a Decreasing APP expression

Molecular Medicine Today

Barrow and Zagorski demonstrated that Ab incubated at different pHs orin distinct solvents shows either mainly random coil/a-helical or b-sheetsecondary structure20. Under the conditions in which the peptide adoptsthe random coil/a-helical structure, it aggregates slowly, but when Abadopts the b-sheetconformation, it aggregates rapidly20–22. By usingsynthetic analogs of Ab (1–40) that contain single amino-acid substitu-tions, we found that a transition between a random coil/a-helix structureand a b-strand conformation in the Ab N-terminal domain modulatesamyloid formation, rendering two different species of Ab in solution:one ‘able’ and another one ‘unable’ to form amyloid21,22 (Fig. 1).Therefore, it seems likely that a key event in amyloidogenesis is the con-version of the normal soluble Ab conformer into the b-sheet-rich amy-loidogenic intermediate (Fig. 1). The available evidence indicates thatthis conversion might be catalyzed by several different factors, including:metal ions (Al31, Zn21), pathological chaperone proteins (apolipoproteinE, amyloid-P component), pH changes, oxidative stress and increasedAb concentration8,14,15,20,22. The emerging picture from studies with syn-thetic peptides is that Ab amyloid formation isdependent on hydrophobic interactions amongaltered Ab peptides that adopt an antiparallelb-sheetconformation (Fig. 1).

Kinetic studies of Ab fibrillogenesis haveproposed a nucleation-dependent mechanismfor Ab aggregation15,19. In this model, the for-mation of amyloid fibrils requires at least twosteps: nucleation and elongation. Nucleation is a thermodynamically unfavorable process re-quiring the assembly of multiple peptidemonomers to form a nucleus; subsequent addi-tion of monomers to the nucleus elongates thepolymer chain. Nucleation and elongation canbe differentiated by several different propertiesand in vitro models have been developed tostudy each of the processes independently15,23.

Most of the experimental evidence indi-cates that the central hydrophobic region ofAb plays an important role in modulatingamyloid formation14,21–26. The APPmutationsin FAD patients located inside the Ab se-quence are all within this region of the pep-tide6,14. On the other hand, it has been shownthat the length of the C-terminal domain ofAb can influence the rate of Ab assembly byaccelerating nucleus formation19, suggestingthat the C-terminal domain of Ab might playan important role in the modulation of fibril-logenesis. Consistent with this idea, it hasbeen shown that the processing of APP mol-ecules that bear mutations associated withFAD is altered to produce a relative increase inthe long forms of Ab (1–42/43)7. The additionof two or three residues at the C-terminal endof Ab1–40 not only increases peptide hy-drophobicity18 but also enhances the stabilityof the abnormal b-sheetconformation in Ab(Ref. 22). In summary, both the central and C-terminal domains of Ab appear to be crucialfor amyloid formation, and the basic unit forfibrillogenesis might be the Ab conformer

adopting an antiparallel b sheetthat consists of strands involving theregions 12–24 and 28–40/42 (Fig. 1). Amyloid formation would pro-ceed by intermolecular interactions between the b-strands of severalmonomers to form an oligomeric b-sheet structure (nucleus orprotofibril) precursor of the amyloid fibrils.

Inhibitors of amyloid formationSeveral, unrelated small molecules (Fig. 3) have been shown to preventAb fibrillogenesis or inhibit Ab toxicity, or both,in vitro. One strategythat has been used to identify lead candidates of amyloid inhibitors isthe screening of large libraries of chemical compounds. The cationicsurfactant hexadecyl-N-methylpiperidinium bromide was found in thisway to be a specific inhibitor of Ab aggregation, possibly by binding toa site on Ab that is necessary for self-assembly27. Sulfonated dyes, suchas Congo red16, small sulfonated anions28 and benzofuran-based com-pounds (Fig. 3)29 have also been described as inhibitors of amyloid for-mation or neurotoxicity associated with amyloid fibrils, or both. Other

346


Figure 3. Chemical structure of some amyloid inhibitors. Several structurally diverse, small chemical mol-ecules have been reported to inhibit either amyloid fibril formation or amyloid neurotoxicity in vitro.Unfortunately, their lack of specificity and, in many cases, their toxicity diminish their therapeutic use.

N N N CH3

NH2 NH2

SO2Na Congo red

Rifampicin

Estrogen

Benzofuran (SKF-74652)

Hexadecyl-N-methylpiperidinium

Anthracycline 4'-iodo-4'-deoxydorubicin


Melatonin

SO2Na

N N

NH

OH

CH3

HO

N N +�N CH3

CH3

OCH3 OH

NH2

OHCH3

CH3

OH

HO

CH3 CH3

OHOH

OHCH2OH

OH

O

O O

O

O

I

CH3

H3C

H3C

H3CO

H3CCOO O

OO

O C�H

N

H

H

N

O

CH3H3CO

+�N

N

O

OCH3H

OMe

NEt2

NicotineO

CI

molecules have been studied for potential inhibitory activity based onprevious reports that indirectly suggested that these compounds couldinteract with amyloid or alter amyloidosis. For example, rifampicinwas studied for amyloid inhibitory activity based on the observationthat elderly leprosy patients who were treated with rifampicin showedan unusual absence of senile plaques in their brains compared with age-matched controls30. In a similar way, various small molecules wereidentified as inhibitors of Ab aggregation or Ab neurotoxicity, includ-ing melatonin31, nicotine32, estrogen33 and anthracycline 49-iodo-49-de-oxydoxorubicin34 (Fig. 3). The usefulness of these small molecules asamyloid inhibitors is compromised by their lack of specificity and theirunknown mechanism of action, which makes it difficult to improvethem. In addition, many of these compounds are highly toxic.

Several proteins have also been described as in vitro inhibitors ofamyloid formation and neurotoxicity, including apolipoproteins E andJ (apoE and J),a1-antichymotrypsin (ACT), serum amyloid P (SAP),laminin, transthyretin and proteoglycans (see Refs 35,36). These pro-teins are co-deposited with Ab in senile plaques8 and bind to Ab invitro37. However, promotion of Ab fibrillogenesis has also been re-ported for most of these proteins, with the exception of apoJ andlaminin (reviewed in Ref. 35). These controversial results suggest thatin vitro assays of Ab fibrillogenesis are of limited value to evaluate theactivity of potential anti-amyloid drugs. Moreover, the only protein forwhich a role in Ab fibrillogenesis has been also studied in vivois apoE,which was shown to enhance amyloid formation. By using APPtrans-genic mice, in which apoE has been knocked out, Bales and colleagues showed a prominent reduction of amyloid-b deposits, andespecially of thioflavine-S-positive plaques38, suggesting that the presence of apoE is essential for amyloid formation.

A more rational approach to make inhibitors of amyloid formationis to design specific peptide ligands based on the well-known self-recognition ability of Ab and on the study of the structural requirementsfor Ab fibrillogenesis. Alanine-scanning studies revealed that the cen-tral hydrophobic region of Ab (residues 16–20) is critical for the inter-action of Ab monomers25. This knowledge was used to develop a pep-tide that contains such a sequence, which binds Ab and partiallyinhibits amyloid fibril formation in vitro25. However, Ghanta and col-leagues showed that binding of a synthetic peptide to Ab was notenough to induce stable inhibition of Ab fibrillogenesis. In their expe-rience, incorporation of a disrupting element was needed in addition tothe fragment that acts in Ab recognition39. A prototype inhibitor thatcomprises residues 15–25 of Ab (recognition element) linked to anoligolysine fragment (disrupting element) prevented Ab toxicity in cellcultures and changed the morphology of amyloid fibrils39. However,the peptide did not alter Ab secondary structure or prevent its aggrega-tion.

b-sheet breaker peptides as a novel strategy to inhibitAlzheimer’s amyloid formationThe formation of amyloid is basically a problem of protein folding,whereby a mainly random-coil soluble peptide becomes aggregated,adopting a b-pleated sheet conformation40–42. Amyloid formation pro-ceeds by hydrophobic interactions among conformationally altered Ab amyloidogenic intermediates (Fig. 1), which become structurallyorganized into a b-sheet conformation upon peptide interaction.Hydrophobicity appears to be important for inducing the interaction ofthe monomers leading to aggregation, while the b-sheetconformationmight determine the ordering of the aggregates into amyloid fibrils.Our strategy was to separate these two properties in short syntheticpeptides homologous to Ab, which bear a similar degree of hydropho-bicity but a very low propensity to adopt a b-sheetconformation(called b-sheetbreaker peptides)43,44 (Fig. 4). As the pathological Abconformation is stabilized by intermolecular interactions, our approachwas to design a peptide that has the ability to bind specifically to Abto form a complex that stabilizes the physiological conformation anddestabilizes the abnormal conformation of Ab.

Peptide designTo design b-sheetbreaker peptides, we focused on the central hy-drophobic region within the N-terminal domain of Ab, amino acids17–21 (LVFFA) (Fig. 4). Proline residues were introduced into the in-hibitor peptide to block b-sheetformation and charged residues were

347


GlossaryAmyloid Ð A generic term that describes fibrillar aggregates thathave a common structural motif: the b-pleated sheet conformation.These aggregates exhibit specific tinctorial properties, including theability to emit a green birefringent glow after staining with Congored, and the capacity to bind the fluorochrome, thioflavin S. Thereare several human diseases of different etiology characterized bythe extracellular deposition of amyloid. The amyloid fibrils usuallycomprise proteolytic fragments of normal or mutant gene products.So far, there are .16 different proteins involved in amyloid deposi-tion in distinct tissues.

Astrogliosis Ð The proliferation and ramification of glial cells in response to various types of brain damage.

b-sheet Ð b sheets, as well as a helices, are the two types of preva-lent, repetitive, secondary structures in folded proteins. b sheetscomprise alternating peptide pleated strands that are linked by hydrogen bonding between the NH and CO groups of the peptidebond. Whereas in a helices the hydrogen bonds are betweengroups in the same strand, in b sheets the bonds are between onestrand and another. The second b strand can come from a differentregion of the same protein or from a different molecule. Therefore,formation of b sheets can be stabilized by protein oligomerization oraggregation.

Conformers Ð Peptides that have identical primary structures butdiffer in their secondary or tertiary structures.

Gene therapy Ð The use of a gene or cDNA to treat a disease, andincludes the use of antisense oligonucleotides (nucleotide se-quences that are complementary to a specific mRNA and down-regulate its expression) and ribozymes (RNA molecules that haveenzymatic activity).

Knockout or null animals Ð Animals with a targeted disruption of aspecific gene by homologous recombination.

Neuritic dystrophy Ð Degenerative nerve terminals.

Neurofibrillary tangles Ð Aggregates that accumulate in the neur-onal bodies and neurites that are composed of a hyperphosphoryl-ated version of the cytoskeletal protein, Tau, and which are organ-ized in the form of paired helical filaments.

added at the ends of the peptide to increasesolubility. Proline was chosen to block b-sheetformation because it rarely forms partof this conformation and does not occur in theinterior of antiparallel b-sheets45. Moreover,the introduction of proline residues into shortpeptides homologous to Ab results in non-amyloidogenic analogues24.

Peptide efficacyb-sheetbreaker peptides of 11 and 5 residues,termed iAb11 and iAb5, respectively (Fig. 4),bind Ab with high affinity, inhibit peptideconformational changes that result in amyloidformation and also dissolve preformed fibrilsin vitro43,44. The 5-residue peptide can alsoprevent neuronal death induced by the forma-tion of b-sheet-rich oligomeric Ab structuresin cell culture experiments44. Furthermore, byusing a rat model of amyloidosis induced byintracerebral injection of Ab1–42, we haveshown that co-injections of the 5-residue b-sheetbreaker peptide decrease cerebral Ab

accumulation and completely block the deposition of fibrillar amy-loid-like lesions in the rat brain44. This is the first study that shows acompound that prevents amyloid-like deposition in vivo. Finally, the b-sheetbreaker peptide injected 8 d after the injection of Ab can disas-semble preformed Ab fibrils in rat brain in vivo, leading to a reductionin the size of the amyloid deposits (E.M. Sigurdsson, B. Frangione andC. Soto, unpublished). No effect was observed by injecting control un-related peptides. Interestingly, removal of amyloid by the b-sheetbreaker peptide reversed the associated histological changes, includingneuronal shrinkage and microglial activation, suggesting that amyloidis responsible for those brain abnormalities. We have also chemicallymodified the 11-residue b-sheetbreaker peptide to significantly in-crease its permeability at the blood–brain barrier and its resistance toproteolysis in plasma without reducing its ability to prevent fibrilloge-nesis in vitro46.

Mechanism of actionThe mechanism by which b-sheetbreaker peptides inhibit and reversefibril formation is unknown. Molecular modeling studies using biasedprobability Monte Carlo simulations and local energy minimization47

suggest that iAb5 interacts in an antiparallel way with the middle region of Ab (Fig. 5). To model the interaction between the b-sheetbreaker peptide and the monomeric a-helical/random-coil Ab con-former , the three-dimensional structure of Ab1–40 in 40% trifluo-roethanol (as reported by Strich and collaborators48) was used. Putativebinding of the b-sheetbreaker peptide to a hydrophobic pocket in thecentral domain of Ab seems to stabilize the physiological conformationof the peptide and prevent it from switching to the b-sheetform (Fig.5a). The b-sheet-rich pathological Ab conformer appears to beformed by two antiparallel b-strands involving the fragments 12–24and 28–40 of Ab with a b-turn around the region 25–27, in agreementwith previously published data (Refs 14,15 and Refs therein) (Fig. 5b).This conformer is most probably unstable in aqueous solution becausehydrophobic residues are exposed to the solvent and become stabilizedby aggregation forming b-sheetoligomers and finally amyloid fibrils.

348


Figure 5. Molecular modeling of the interaction between the 5-residue b-sheet breaker peptide (iAb5) and dif-ferent amyloid-b peptide (Ab) conformers. The three-dimensional structure of Ab1Ð40 in 40% trifluoroethanolelucidated by nuclear magnetic resonance (NMR)48 was used to model the interaction with iAb5. The struc-ture of iAb5 was estimated by using the ICM software47, and is shown in the ballÐstick form with the residueslabeled as follows: Leu1, blue; Pro2, red; Phe3, white; Phe4, green and Asp5, purple. (a) Docking studies sug-gest that iAb5 binds to a hydrophobic pocket formed by the Ab residues Leu17, Phe20, Ala21, Ala30, Ile31and Ile32 (magenta). (b) The b-sheet-rich pathologic Ab conformer was modeled by energy minimization afterfitting the fragments 12Ð24 and 28Ð40 of Ab to b-strand structures. The inhibitor peptide might bind to the re-gion 13Ð19 of b-sheet-rich Ab. Leu1 and Phe3 of iAb5 might bind hydrophobically to Leu17 and Phe19 of Ab,and Asp5 of iAb. For simplicity, the structure of the amyloidogenic Ab conformer is represented in themonomeric form. However, it is likely that this conformer adopts mainly a dimeric or oligomeric form.

N

N

a b

C

C

Figure 4. b-sheet breaker peptides as a novel strategy to inhibit and reverseamyloidosis in AD. (a) Several b-sheet breaker peptides have been designedbased on the hypothesis that amyloid deposition could be inhibited by shortsynthetic peptides partially homologous to the amyloid-b peptide (Ab) regionthat undergoes abnormal conformational change to give rise to the amyloido-genic Ab intermediate. b-sheet breaker peptides contain residues that stabilizethe normal conformation of Ab, thus disrupting b-sheet formation.

sAb Conformationally�altered Ab

APP


DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVAb1Ð40

iAb11 RDLPFFPVPIDiAb9 RDLPFFPVDiAb7 LPFFPVDiAb6 LPFFVDiAb5 LPFFDiAb4 LPFFiAb3 PFF

b-sheet breaker peptides

b-sheet breaker peptides

a

b

The modeling studies suggest that the inhibitor peptide could bind toresidues 13–19 of Ab, mimicking the interaction between b strands ofadjacent monomers (Fig. 5b). However, the backbone constraints imposed by the proline residue determine that the inhibitor peptidedoes not fit in the b sheet, displacing some of Ab residues and induc-ing the disruption of the b-sheetstructure.

Although the theoretical model accounts for some experimental results, its relevance is uncertain before experimental evaluation.Indeed, many aspects of the model are purely theoretical, owing tothe limited high-resolution structural data available for Ab. The ten-dency of the peptide to aggregate under the conditions used for struc-tural studies and the non-crystalline nature of the aggregates have pre-cluded accumulation of detailed structural information14,15,23. Indeed,no crystallographic data are available and the nuclear magnetic resonance (NMR) studies using full-length Ab have been done in thepresence of organic solvents or detergents that minimize peptide aggregation, but also alter Ab structure15,20,48. The relevance of thesefindings is unclear. In fact, studies of short Ab fragments under morephysiological conditions, using solid-state or solution NMR, havereached different conclusions49,50.

Beyond Alzheimer’s diseaseThe concept of b-sheetbreaker peptides provides a potentially generalstrategy to treat diseases caused by defective protein folding, such asamyloidosis-related disorders, prion diseases, disorders associatedwith serpin deficiency, Parkinson’s disease, amyotrophic lateral scle-rosis and Huntington’s disease (reviewed in Refs 40–42). The commonmechanism in those diseases is the abnormal folding of a natural pro-tein into a pathological conformer that becomes aggregated or ex-hibits toxic activity. b-sheet breaker peptides that combine a se-quence similarity to the region of the protein involved in the abnormalfolding and the ability to block the pathological conformationalchanges offer the possibility of generating therapeutic compounds specifically directed at each of these diseases. We haverecently applied this approach to develop short b-sheetbreaker pep-tides that prevent and reverse the prion protein conformationalchanges implicated in the pathogenesis of spongiform en-cephalopathies (C. Soto et al., unpublished). We hope that b-sheetbreaker peptides, or non-peptidic derivatives of them, will prove to beuseful therapeutic agents to prevent or retard amyloidogenesis in ADand possibly other diseases in which the aggregation of b-sheet-con-taining peptides contributes to their pathology.

Acknowledgments.I thank E. Sigurdsson, G. Saborio, A. Kumar and B. Frangione from New

York University School of Medicine and E. Castaño and L. Morelli from University ofBuenos Aires for collaboration in the study of b-sheet breaker peptides. This study was sup-ported by NIH (MH56472), Fundación Andes of Chile and AXONYX, Inc.

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The outstanding questions

• Is amyloid plaque formation a central and triggering factorof Alzheimer’s disease (AD) pathogenesis?

• Why does soluble amyloid-b peptide (Ab) becomedeposited in insoluble amyloid plaques?

• Which is toxic to the human brain:amyloid deposits orsoluble altered Ab peptides?

• Can b-sheetbreaker peptides inhibit and reverse plaquedeposition in the brains of AD patients?

• Is amyloid inhibition a viable therapy for AD?

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350


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Plaque busters: strategies to inhibit amyloid formation in Alzheimer's disease

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