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Contents lists available at ScienceDirect

Peptides

journa l homepage: www.e lsev ier .com/ locate /pept ides

evelopment of retro-inverso peptides as anti-aggregation drugs for �-amyloidn Alzheimer’s disease

alpreet Matharua, Omar El-Agnafb, Amna Razvia, Brian M. Austena,∗

Neurodegeneration Unit, Basic Medical Sciences, St George’s, University of London, Cranmer Terrace, London SW17 0RE, UKDepartment of Biochemistry, Faculty of Medicine and Health Sciences, United Arab Emirates University, P.O. Box 17666 Al-Ain, United Arab Emirates

r t i c l e i n f o

rticle history:eceived 13 April 2010

a b s t r a c t

Alzheimer’s disease (AD) is a devastating degenerative disorder of the brain for which there is no cureor effective treatment. There is much evidence to suggest that �-amyloid protein (A�) aggregation in

eceived in revised form 29 June 2010ccepted 29 June 2010vailable online xxx

eywords:-Amyloid

the brain leading to deposits is an important step in the development of AD. Recently, two peptides,RGKLVFFGR (OR1) and RGKLVFFGR–NH2 (OR2) containing the sequence KLVFF, which is the central region(residues 16–20) of A�, have been found to be potent inhibitors of A� aggregate formation. Here we reportthat retro-inversion of these sequences increases efficacy of the peptides in the inhibition of aggregationand toxicity of �-amyloid. We describe the synthesis and inhibitory properties of these retro-inverso

lzheimer’s diseaseggregation

peptides.

. Introduction

There is considerable accumulated evidence that the depositionf �-amyloid protein (A�) in the brain is a cause of the cogni-ive decline seen in Alzheimer’s disease (AD). The aggregation of

onomeric A� into larger oligomeric and fibrillar forms is seen asn important step in the development of pathology and cognitiveoss. It seems increasingly likely that early, soluble oligomers arectually the toxic species responsible for neurodegeneration andeuronal cell death [18]. The soluble oligomeric A� is now known tolock synaptic plasticity required for memory formation, particu-

arly in the hippocampus, where neuronal loss is seen as the diseaserogresses [18]. These findings have lead to the hypothesis thatmall molecules which can block, slow down or reverse A� aggre-ation, particularly in its early stages, might provide an attractiveherapeutic approach for targeting the underlying disease progres-

Please cite this article in press as: Matharu B, et al. Development of reAlzheimer’s disease. Peptides (2010), doi:10.1016/j.peptides.2010.06.

ion of AD. It has been shown in vitro that synthetic A� peptideeadily aggregates from solution into fibrils with a crossed �-sheetonformation. Coincident with the conversion of monomeric A�o the fibrillar forms is a transition from random coil to �-sheet

Abbreviations: A�, amyloid peptide; AD, Alzheimer’s disease; ELISA, enzyme-inked immunosorbent assay; MTT, methylthiazoletetrazolium; PBS, phosphateuffered saline; Tris, Tris(hydroxymethyl)amino-methane; LDH, lactate dehydro-enase; BSA, bovine serum albumin; TMB, 3,3,5,5-tetramethylbenzidine; BBB,lood–brain barrier.∗ Corresponding author. Tel.: +44 2087255651; fax: +44 2087255651.

E-mail addresses: Balpreet@doctors.org.uk (B. Matharu),gk200@sgul.ac.uk (B.M. Austen).

196-9781/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.peptides.2010.06.033

© 2010 Elsevier Inc. All rights reserved.

structure [16]. A� is amphiphilic, with a hydrophilic N-terminusand hydrophobic C-terminus; the length of the latter affects therate of aggregate formation [4]. The short hydrophobic stretch atresidues 16–20 (the sequence KLVFF) in the central portion of A�appears to be critical for its aggregation [17]. The fact that A� is anatively unfolded peptide, which has little or no ordered structureunder normal physiological conditions, makes the rational designof compounds that can stabilize the native, non-toxic conformationof A� a challenging task. However, researchers have been success-fully able to design small modified synthetic peptides based onthe hydrophobic central region (residues 16–20) of A� that canprevent its aggregation. Recently, we have designed peptide-basedinhibitors (named OR inhibitors) of A� aggregation and toxicity [2].Our findings have led us to hypothesize that, the OR peptides rep-resent the starting point for designing peptide inhibitors that aresuitable for chronic therapy and might be used as new drugs forthe treatment of AD. Here we describe retro-inversion of the bind-ing sequence, as this has been discovered in other systems to leadto peptides which are stabile to peptidases in cells, and in whichrelative orientations of the amino acid side chains are the same asthose in the original sequence [8]. A preliminary report of theseretro-inverso inhibitors has been published [1].

2. Methods

tro-inverso peptides as anti-aggregation drugs for �-amyloid in033

2.1. Peptide synthesis, purification and characterization

A�40 and 42, were synthesized and purified by HPLC asdescribed [4]. Monomeric A�40 and A�42 [5] were prepared

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y triple recycling in TFA and 1,1,1,3,3,3-hexafluoroisopropanolHFIP), and drying under a slow stream of dry N2. Dissolution of thearget to 200 �M was with sterile distilled water. Aliquots (200 �lach) were stored at −70 ◦C until used.

In the following nomenclature, small case letters representmino acid residues in d-configuration. The peptide rGffvlkGr–NH2ontaining the retro-inversion of OR2 sequence was synthe-ized on Fmoc PAL–PEG–polystyrene resin (Applied Biosystems)0.2 mmoles; 0.22 mmole/g), coupling in succession, Fmoc-d-rg(Pbf), Gly, d-Lys(OtBu), d-Leu, d-Val, d-Phe, d-Phe, Gly and-Arg(Pbf), with PyBOP or HATU. Other peptides were synthesizeds described previously [2]. rGffvlkGr-1,5-diaminopentane wasynthesized by reductive amination of formyl polystyrene resin-{4-formyl-3-methoxyphenoxy}butyric acid (FMBP) NovaGel TMith mono-1-butoxycarbonyl, 1,5-diminopentane (Tosyl salt)

Novabiochem), and then by Fmoc peptide synthesis. LPFFD [14]as synthesized on Fmoc Asp(tBu)–PEG–polystyrene, couplingith Fmoc l amino acids. N-terminal acetylation was achieved

y treatment of the peptidyl resin with N-acetylimidazole in 5 Mxcess. The target peptides were cleaved and deprotected in TFA,riisopropylsilane (5%) and water (5%), triturated in ether, and puri-ed by HPLC on a column of Dynamax C8, in 0.1% TFA with ancetonitrile gradient. Purity was verified by MALDI mass spec-rometry on a Shimidazu-Kratos Axima MALDI mass spectrometer.eptides were incubated together with A� in sterile 0.1 M Tris–HClpH 7.4), to test inhibition of the formation of oligomers and aggre-ates.

.2. ELISA to measure Aˇ oligomers

A novel ELISA has been developed [10,13] which detects A�ligomers. Solutions of A� rapidly yield soluble oligomers in aoncentration-dependent manner, which are detected by the ELISA.he ELISA uses the same antibody for capture and detection in aandwich format which allows the assay to differentiate oligomersrom monomers, as monomers have only one epitope, and doot bind the biotinylated form of the antibody while the epitope

s occupied with the non-biotinylated antibody. Insoluble fibrilsave a reduced response as the epitopes are occluded [10,13]. TheLISAs were performed on 96-well immunoassay plates (Nunc,alge Europe Ltd., Hereford, UK) and were used to measure thefficacy of the inhibitors at preventing A� oligomerization. All dilu-ions and wash steps, unless otherwise stated, were made usinglocking buffer (phosphate buffered saline (PBS), 1% gelatin, 1%ovine serum albumin (BSA), 0.05% Tween 20).

Lyophilized synthetic A�40 and A�42 were dissolved to 200 �Mn sterile distilled water before dilution to a final concentration of0 �M A� in 10 mM PBS, with and without various concentrationsf the peptide inhibitors. These solutions were then incubated for4 h at 37 ◦C and were then further diluted to a working concentra-ion of 1 �M of A� in blocking buffer before addition to the blockedlates. 96-Well ELISA plates (Greiner Bio-One Inc., Longwood, FL,SA) were coated overnight at 4 ◦C with 100 �l/well rabbit capturentibody anti-NTA4 [15] diluted in PBS (pH 7.4) to 3 �g/ml. Afterntibody removal, the plates were blocked using 100 �l per welllocking buffer and then incubated for 1 h at 37 ◦C. After blockinguffer removal, 100 �l of the A� solutions were added to each wellnd incubated for 2 h at 37 ◦C. After four washes, 3 �g/ml of biotiny-ated anti-NTA4, was added in 100 �l/well and incubated for 1.5 ht 37 ◦C. After four washes, Extravidin-peroxidase (Sigma–Aldrich)1:1000 dilution in blocking buffer) was added (100 �l/well) and

Please cite this article in press as: Matharu B, et al. Development of reAlzheimer’s disease. Peptides (2010), doi:10.1016/j.peptides.2010.06.

ncubated for 1 h at 37 ◦C. After four washes in PBST, followed by oneash with PBS, 3,3,5,5-tetramethylbenzidine (TMB) (100 �l/well,

uropa Biosciences, Cambridge, UK) was added. Termination ofolor development was by the addition of 25 �l/well 2 M H2SO4nd absorbance at 450 nm was read using a Multiskan Ex microtitre

PRESSxxx (2010) xxx–xxx

plate reader (Thermo Electron Corp.). Results were expressed as %absorbance compared to the A� peptide without inhibitor.

2.3. Thioflavin binding assay

A�40 was incubated in sterile PBS together with peptideinhibitors at various molar ratios for up to 12 days at 37 ◦C. Amyloidfibril formation was monitored by fluorescence after addition ofThioflavin-T (Th-T). Th-T is a fluorescent dye, which interacts withfibrils and oligomers containing crossed �-sheet structure. The pro-cess is accompanied by a characteristic increase in fluorescenceintensity at 480 nm. A 10 �l sample of each peptide solution wasdiluted into 190 �l Th-T (1 mM) in PBS. Fluorescence was measuredin 96-well black plates (Nunc, Denmark) using a Victor microplatereader, with excitation at 440 nm and emission at 480 nm. To allowfor background fluorescence, the fluorescence intensity of a blanksolution (Th-T alone in PBS) was subtracted from all readings.

2.4. Transmission electron microscopy

Electron micrographs were produced from aged A� solutionswith or without inhibitors. The samples were deposited on Formvarcoated 400 mesh copper grids, fixed with 0.5% (v/v) glutaraldehyde,negatively stained with uranyl acetate, and examined on a JEOLJEM-1010 transmission electron microscope.

2.5. Resonant acoustic profiling

Binding of peptides to immobilized A� was measured byresonant acoustic profiling on a RAP4 biosensor (Akubio Ltd., Cam-bridge, UK) [7]. Incubated A�40 or A�42 were immobilized at50 �g/ml on a carboxylate surface by treatment with EDC and NHSin 10 mM sodium acetate (pH 4.5), with mouse IgG (50 �g/ml) ona reference channel as a control surface. Peptide inhibitors at vari-ous concentrations in PBS were injected over all channels for 60 s,followed by a 180 s dissociation period. A 30 s injection of 100 mMglycine (pH 1.5) was used to regenerate the surface.

2.6. Tissue culture of SHSY-5Y human neuroblastoma cells

Human SHSY-5Y neuroblastoma cells (European Collection ofCell Cultures, Porton down, Salisbury, Wilts, UK) were culturedin Dulbecco’s minimal essential medium (MEM)/nutrient mix F-12 (1:1, v/v) (GibcoTM, Invitrogen, Paisley, UK) supplemented with10 i.u. of penicillin/ml and 100 �g/ml streptomycin (GibcoTM, Invit-rogen, Paisley, UK), 15% v/v fetal calf serum, 1% v/v non-essentialamino acids (GibcoTM, Invitrogen, Paisley, UK), and 2 mM glutamine(GibcoTM, Invitrogen, Paisley, UK). The cells were routinely subcul-tured once every 7 days when confluent at a dilution of 1:10, andgrown at 37 ◦C in a humidified incubator with 95% air/5% CO2 in75 cm2 tissue culture flasks.

2.7. Measurement of cell viability

SHSY-5Y cells were plated at 7500 cells/well (100 �l/well)in 96-well plates (Greiner Bio-One Inc., Longwood, FL, USA) infull medium and incubated overnight at 37 ◦C. Following a PBSwash, a suspension of aged A�40 (25 �M; 100 �l/well), containingdifferent concentrations of inhibitors, all in OPTI-MEM serum-free media (GibcoTM, Invitrogen, UK) were added to the cells

tro-inverso peptides as anti-aggregation drugs for �-amyloid in033

and incubated for another 48 h at 37 ◦C. The cells were washedwith 100 �l/well PBS prior to the addition of 0.6 mg/ml MTT(3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide) infull medium (100 �l/well). Following incubation for 4.5 h at 37 ◦C,50 �l of MTT solution was removed from each well and replaced

IN PRESSP

ptides xxx (2010) xxx–xxx 3

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Fig. 1. The retro-inverso peptide, rGffvlkGr-1,5-diamino pentane (clear bars) andthe peptides LPFFD (grey bars) or acetyl-QKLVFF-amide (black bars) were shownto inhibit A�42 oligomerization, as measured by ELISA. HFIP/TFA-treated A�42 at

stunted fibrils, which were reduced in number at higher concentra-tions of rGffvlkGr–NH2 (25 �M) (Fig. 4). In contrast, the absence ofrGffvlkGr–NH2, or the presence of KLVFF–NH2 at 200 �M yielded amesh of long, intertwined fibrils to be formed from A�. The pres-

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ith 150 �l of lysis buffer (containing 20% w/v SDS and 50% v/v N-N-imethyformamide, pH 4.7). Plates were then incubated overnightt 37 ◦C. Absorbance readings were taken at 570 nm using a Multi-kan Ex microtitre plate reader (Thermo Electron Corp.). Data arexpressed as % MTT reduction compared to a 100% signal fromntreated cells.

.8. LDH toxicity assay

SHSY-5Y cells were plated at 7500 cells/well (100 �l/well) in6-well plates (Greiner Bio-One Inc., Longwood, FL, USA) in fulledium and incubated overnight at 37 ◦C. After a 100 �l/well PBSash, 100 �l/well of A� solutions, containing various concentra-

ions of aggregation inhibitors in OPTI-MEM serum-free mediaGibcoTM, Invitrogen, UK) were added to the plate and then incu-ated at 37 ◦C for another 48 h. The plate was centrifuged at 500 rpmt 25 ◦C for 10 min, and 50 �l of each supernatant transferred tosecond 96-well plate. Following incubation, the LDH assay was

ompleted using a Cytotoxicity Detection Kit TOX7 (Sigma–Aldrich,oole, Dorset) in which LDH reduces NAD+, which then convertstetrazolium dye to a soluble, colored formazan derivative. The

ssay substrate, co-factor solution and dye solutions purchasedere mixed together in equal volumes, and 100 �l, added to each

0 �l of supernatant, incubated at 37 ◦C for 30 min, and read at90 nm. Data is expressed as % cell death compared to the totalDH released by cell lysis in 0.1% Triton X-100.

. Results

We have recently reported two peptides, RGKLVFFGR (OR1)nd RGKLVFFGR–NH2 (OR2) that are effective inhibitors of fibrilormation from A�, while only OR2 also inhibits oligomer forma-ion and A�-induced toxicity towards neuronal cells in culture2]. Our aim in this study is to design peptides based on theR2 sequence with increased efficacy as inhibitor of A� aggre-ation and toxicity, and stable for chronic treatments in vivo.his was achieved by incorporating d-amino acids and reversinghe sequence of OR2 peptide. Retro-inverso versions of OR2 pep-ides, i.e. rGffvlkGr–NH2 and rGffvlkGr-1,5-diaminopentane wereynthesized, purified and characterized by analytical HPLC andALDI mass spectrometry, and found to be >95% in purity. 1,5-iamopentane was incorporated for future use in penetrating thelood–brain barrier in vivo [9]. They were then compared withGKLVFFGR–NH2 and Ac-QKLVFFG–NH2 as normal sense inhibitorsf A� aggregation, and LPFFD as a �-sheet breaker peptide incor-orating a proline reside into the binding sequence found in A�14] to test their effects on A� early and late aggregate forma-ion using an ELISA, thioflavin-T binding and electron microscopy,nd on A� toxicity to human SHSY-5Y neuronal cell lineultures.

.1. Inhibition of Aˇ oligomer formation

An ELISA was developed previously in our laboratory thatetects only soluble oligomers of A�, and does not recognize theonomeric forms of A�, whilst the fibrillar forms of A� give a much

educed response [10,13]. The retro-inverso peptide rGffvlkGr-1,5-iamino pentane was found to be more effective than LPFFD orc-QKLVFFNH2 at inhibiting A�42 oligomer formation (Fig. 1).

Please cite this article in press as: Matharu B, et al. Development of reAlzheimer’s disease. Peptides (2010), doi:10.1016/j.peptides.2010.06.

etro-inverso peptides, rGffvlkGr–NH2, or rGffvlkGr-1,5-diaminoentane at 25 �M reduced A�40 aggregation to ∼3%, whereasLVFF–NH2 at 25 �M inhibited A�40 oligomer formation by only0% of that seen in the absence of inhibitor (Fig. 2). Inhibition was

ncreased by concentrations of inhibitors up to 100 �M.

50 �M, in PBS at pH 7.4 together with peptide inhibitors, were incubated for 24 hat 37 ◦C. After dilution 1–50 in blocking buffer, the extent of oligomer formationwas measured by ELISA, with the absorbance obtained by incubation of A�42 in theabsence of any inhibitor taken as 100%.

3.2. Inhibition of fibril formation

Fibril formation of A� (50 �M) was measured by Th-T bindingto aggregates containing �-sheets after incubation of A� solutionin the presence of the peptide inhibitors for up to 12 days (Fig. 3).Peptide RGKLVFFGR at 1:2 molar ratio with A� (100 �M) allowedfibrils to form after 12 days, as did KLVFF–NH2. In contrast nei-ther rGffvlkGR–NH2 nor RGKLVFFGR–NH2 (OR2) allowed fibril orprotofibril formation (Fig. 3). KLVFF–NH2 allowed fibrils to form at2:1 1:1 and 1:2 molar ratios, and there was a significant increasein fibril formation after 12 days. Fibril formation in A� (100 �M),was also visualized by EM. Co-incubation with rGffvlkGr–NH2 at16.7 �M led to the formation of only small numbers of short,

tro-inverso peptides as anti-aggregation drugs for �-amyloid in033

Fig. 2. The retoinverso peptides: rGffvlkGrG-1,5-diamino pentane (grey bars),AcrGffvlkGr–NH2 (clear bars) and KLVFF–NH2 (black bars) were incubated withHFIP/TFA-treated A�40 (50 �M) in PBS at pH 7.4 for 24 h. Samples were diluted 1–50in blocking buffer, and oligomers measured by ELISA. Plotted is the OD obtained asa % of that obtained by A�40 in the absence of inhibitors.

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F oflavin3 d at 4

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ig. 3. The effect of peptide inhibitors on A� 1–40 fibrilization as measured by thi7 ◦C for up to 12 days, and fibril formation was measured by fluorescence produce

nce of high concentrations of RGKLVFFGR (OR1) stopped A�40onverting into fibrils, although a small number of protofibrilsere seen. As reported previously [2], the effect of the addition

f the C-terminal amide in is to prevent oligomer formation fromonomers, whereas the retro-inversion increases efficacy.

.3. Binding of inhibitor peptides to Aˇ

A�40 and A�42 were immobilized to a RAP Akubio carboxy-

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ate surface activated as N-hydroxysuccinimide esters with ethyliethyl carbodiimide. rGffvlkr–NH2 and QKLVFFG–NH2 at five dif-erent concentrations were injected over the A� surfaces (withmmobilized mIgG in the control cell monitored simultaneously).he acoustic resonance responses obtain on the A� surfaces at 25 ◦C

ig. 4. Electron microscopic examination of the effect of peptides on A�40 fibril formatiooncentrations of inhibitors. The figure shows negatively stained samples. Marker is 100

-T binding assay. Peptide inhibitors were incubated with A�40 (50 �M) in PBS at80 nm after addition of thioflavin-T (1 mM).

in PBS, with the control mIgG response subtracted, are shown inFig. 5. Kinetic parameters were derived from curve fitting to Lang-muir binding models:

A� aggregates Inhibitor ka (M−1 s−1) kd (s−1) KD

A�40 rGffvlkGr–NH2 3.3 × 104 1.6 × 10−3 49.4 nMA�42 rGffvlkGr–NH2 3.7 × 104 1.6 × 10−3 43 nMA�40 QKLVFFG–NH2 838.9 6.9 × 10−3 82.4 �MA�42 QKLVFFG–NH2 598.5 4.5 × 10−3 7.5 �M

tro-inverso peptides as anti-aggregation drugs for ˇ-amyloid in033

The results showed that retro-inverting the binding sequence inthe peptides increased the association rate to A� by about 50-foldand affinity for A� about 500-fold, and decreased the dissociationrates 4-fold.

n A�40 (100 �M) in PBS were incubated either alone of in the presence of variousnm.

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F resonc 4.5), ar er allg RAP4

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ig. 5. Binding of inhibitor peptides to immobilized aggregated A� measured byarboxylate surface by treatment with EDC and NHS in 10 mM sodium acetate (pHGffvlkr–NH2, or Ac-QKLVFFG–NH2 at various concentrations in PBS were injected ovlycine (pH 1.5) was used to regenerate the surface. Responses were measured in a

.4. Inhibition of Aˇ40 toxicity to neuronal cells in culture

Toxicity of aged A� solutions in the presence of inhibitors waseasured using SHSY-5Y human neuroblastoma cells in culture

Fig. 6). Residual redox activity in living cells was measured byptake and reduction of the dye MTT, and cell death by releasef lactate dehydrogenase (LDH). Although the MTT assay dependsn active mitochondria, and requires A� to be taken up into cells10], it has been widely used as a measure of cell death, and wasound to inversely correlate with the extent of LDH release in theresent study. rGffvlkGr-1′5-diamino-pentane inhibited the toxic-

ty of aged A�40 solutions in a concentration-dependent manneretween 25 and 100 �M (p < 0.001 at all concentrations). It wasffective at both high and low concentrations showing 57% effi-acy at 25 �M (p < 0.001), with greatest efficacy at 100 �M (99%nhibition) (see Fig. 6a). Both acetyl-QKLVFF-amide and LPFFD alsohowed concentration-dependent inhibition of A�40 toxicity, butere both less effective against A�40 toxicity than rGffvlkGr-

,5-diamino pentane, which showed >90% inhibition of toxicityp < 0.001) at all concentrations tested (Fig. 6a). rGffvlkGr–NH2 waslso effective against the toxicity imposed by aggregated A�42 at5 �M, with complete protection shown at 50 �M, whereas thether inhibitors without retorinversion were relatively ineffectiveFig. 6b).

The inhibitors were effective at inhibiting cell death mea-ured by LDH release from SHSY-5Y cells treated with aggregated�1–42 (Fig. 7a) and aggregated A�40 (Fig. 7b). KLVFF–NH2 wasost effective at 10 �M and less protective at lower and higher

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oncentrations, possibly due to the high propensity of A�1–42o aggregate, compared to A�1–40. In contrast the retro-inversoGffvlkGr–NH2 showed increased inhibition up to 96% at 100 �M,here it reduced the LDH release by 16-fold compared to cells incu-

ated without inhibitor. The retro-inverso rGffvlkGr-1,5-diamino

ant acoustic profiling. Aged A�40 or A�42 were immobilized at 50 �g/ml on and mouse IgG (50 �g/ml) was layered on a reference channel as a control surface.channels for 60 s, followed by a 180 s dissociation period. A 30 s injection of 100 mMAkubio acoustic resonance biosensor.

pentane also showed maximum inhibition of 86% (p < 0.001) at25 �M, but less protection at higher concentrations. The peptideinhibitors were less effective at reducing the LDH release by A�42than A�40 (Fig. 7b), although both KLVFF–NH2 and rGffvlkGr–NH2were inhibitory at 25 �M.

4. Discussion

Convergent pathological, biochemical, animal models andgenetic evidence suggests that the formation of A� deposits is animportant and, probably, seminal step in the development of AD[18]. Over the years there have been several attempts to lowerthe cerebral A� burden in AD: these include reducing APP geneexpression; altering APP processing; inhibiting A� fibril formation;accelerating the clearance of amyloid plaques or soluble A� and/orpreventing amyloid neurotoxicity. Also, inhibition of �- and/or �-secretases could decrease A� production. A great deal of efforthas been made in screening and designing compounds that wouldinhibit the aggregation and toxicity of A�. Recent studies supportthe idea that early aggregates or “soluble oligomers” of A� are thetoxic species that drive neurodegeneration and neuronal cell death,rather than mature amyloid fibrils [2]. Considering the importanceof soluble oligomeric forms of A� in AD pathogenesis, it is nowclear that drug development in this area should focus on inhibitorsof A� oligomerization rather than inhibitors of fibril formation.Recently, we have designed peptide-based aggregation inhibitorscontaining the A� amino acid sequences (KLVFF) from part of thebinding region responsible for A� self-association (residues 16–20),

tro-inverso peptides as anti-aggregation drugs for �-amyloid in033

with RG−/−GR residues added at their N- and C-terminal ends toaid solubility. Two such peptides (RGKLVFFGR, named OR1, andRGKLVFFGR–NH2, named OR2) were effective inhibitors of A� fib-ril formation, but only OR2 inhibited A� oligomer formation andneurotoxicity, suggesting a dominant effect of the C-terminal moi-

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Fig. 6. (a) Protection of human SHSY-5Y neuronal cells against 25 �M A� 1–40 induced toxicity. SHSY-5Y cells were incubated with peptide inhibitors LPFFD (black bars),Ac-QKLVFF–NH2 (grey bars) and the retro-inverso peptide rGffvlkGrG-1,5-diamino pentane (clear bars) at various concentrations together with aggregated aged �-amyloid-4 aininp TT cola ationsa

eAbhdt[sdthtpftoocrr

0 (25 �M) for 24 h. Toxicity was measured by the MTT coloration given by the remeptide inhibitors, the cells incubated with only A�40 (25 �M), gave 41% of the Mgainst A�42 (25 �M) for 24 h by peptide inhibitors at 5, 10, 26 and 50 �M concentrnd expressed as a % of MTT color seen in the absence of A�.

ty. Increase in inhibition by incorporation of positively chargedrg residues into these inhibitors may have increased interactiony increasing surface tension [6]. These findings have led us toypothesize that, the OR2 peptide represent the starting point foresigning peptido molecules that are more suitable for chronicherapy and might be used as new drugs for the treatment of AD2]. A potential problem associated with peptide therapy is highensitivity of peptides to proteolytic degradation, however, theseifficulties can be minimized by using d-amino acids. Transporthrough the blood–brain barrier (BBB) is also difficult. Methodsave been developed for the delivery of exogenous peptides acrosshe BBB with the help of membrane-permeable carrier such asolyamines [9]. In this study we have designed peptide compoundsor chronic treatment in vivo based on the structure of OR2 pep-ide, and investigated the effectiveness of these compounds on theligomerization and toxicity of A�. Retro-inverse peptides based

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n OR2 sequence were synthesized using amino acids of the d-onfiguration rather than the l found in the native sequence. Theetro-inverse configuration gives rise to peptides with increasedesistance to proteolytic degradation and turnover in living organ-

g active cells, and expressed as a % of MTT color seen in the absence of A�. With noor obtained in the absence of A�. (b) Protection of human SHSY-5Y neuronal cells. Toxicity was measured by the MTT coloration given by the remaining active cells,

isms. For the peptide inhibitor rGffvlkGr-1,5-diamino pentane, theC-terminal di-amine was incorporated with a view to aiding trans-port across the BBB [9] although in vivo transport was not tested inthe present studies

rGffvlkGr–NH2 peptide proved to be very potent at inhibit-ing aggregation of A�, possibly due to increased interactionsbetween the aromatic side chains of FF in the retro-inverso pep-tide and the native sequence in A�. Inhibition was greater thanthat of RGKLVFFGR–NH2, the recognition sequence in the nat-ural l-configuration. In ELISA’s which measure soluble oligomerformation in A�, inhibition by rGffvlkGrG-1,5-diamino pentanewas total at 100 �M (98%) (Fig. 2). The retro-inverso configurationgives the peptide an increased ability to prevent oligomer forma-tion. The effect of retro-inverting the aggregation domain showsmost clearly in comparison of RGKLVFFGR with rGffvlkGr–NH2. Thelatter was more efficacious in both preventing soluble oligomer

tro-inverso peptides as anti-aggregation drugs for �-amyloid in033

formation, and preventing the production of thioflavin-T-positivefibrils, preventing the elongation of clumps into fibrils seen by EM.These effects suggest that fibrils are formed from monomers by thesame pathway as oligomers. Thus rGffvlkGr–NH2, prevents both

ARTICLE ING ModelPEP-68106; No. of Pages 7

B. Matharu et al. / Peptides

Fig. 7. (a) Protection of SHSY-5Y cells against 50 �M aggregated A� 1–42 inducedtoxicity. SHSY-5Y cells were incubated with aggregated A�42 (50 �M) and retro-inverso inhibitors rGffvlkGr-1,5-diamino pentane (black bars) and rGffvlkGr–NH2

(clear bars) or KLVFF–NH2 (grey bars) for 28 h (p < 0.001 at all concs.). LDH iseXb

otintbctpfftAtrrocgsaci

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xpressed as the % released compared to the total release by cell lysis in 0.1% Triton-100. (b) Protection of SHSY-5Y cells against aggregated A�40 (50 �M) over 48 hy peptide inhibitors.

ligomer and fibril formation. Thus, rGffvlkr–NH2 was more effec-ive at protecting human neuronal cell lines against A� toxicity thannhibitors containing l amino acids (Figs. 6 and 7). However, it wasoted that at 25 �M concentration of A�40 at inhibitor concentra-ion of 100 �M % MTT reduction was increased by 138%. There coulde a number of reasons for this: (a) rGffvlkGr–NH2 may increaseell growth and therefore, there could be increased MTT reduc-ion due to there being more cells; (b) it may be inducing enzymeathways which are involved in the reduction of MTT and there-ore, artificially increasing % MTT reduction. Measure of LDH releaserom SHSY-5Y cells treated with rGffvlkr–NH2 and A�1–42 showedhat LDH decreased in the presence of rGffvlkGr–NH2 (Fig. 7b).ggregated A�40 was less effective at inducing the release of LDH

han A�42, in keeping with the reported reduced toxicity of A�40.GffvlkGr–NH2 proved effective at preventing cell death by A�40,educing LDH released down to 4% of that released by total lysisf the cells (Fig. 7a). Therefore, it is likely that the retro-inverseonfigurations give rise to more potent inhibition of A�40 aggre-ation. Comparison of binding to A� using an acoustic biosensorhowed that retro-inversion increased on-rates and affinity for A�,nd decreased dissociation rates. This would be preferred pharma-okinetics for the target of aggregated A�, if an extended time ofnteraction was required to prevent aggregation.

Dispersal of A� fibrils by inhibitors or immunotherapy [12]ould be a double-edged sword in terms of therapy. Fibrils which

Please cite this article in press as: Matharu B, et al. Development of reAlzheimer’s disease. Peptides (2010), doi:10.1016/j.peptides.2010.06.

re relatively inert could be converted back into soluble oligomersr protofibrils which are more toxic [3]. However, there was novidence that the peptide inhibitors synthesized converted pre-ormed fibrils back to oligomers. In these studies, inhibitors thatere active at preventing oligomer formation (e.g. rGffvlkGr–NH2),

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PRESSxxx (2010) xxx–xxx 7

were also active at inhibiting fibril formation, indicating that fibrilsare likely to be an end-point of the oligomer pathway, so prevent-ing oligomers forming from monomers of A�, will also prevent fibrilformation. The C-terminal amidated versions of the inhibitor pep-tides (e.g. rGffvlkGr–NH2) are preferred, as the carboxy terminalvariants, which prevent fibril formation have less activity than theamidated forms in preventing oligomer formation, On the basisof CD and FT-IR measurements on aggregating solutions of A�40and A�42, we proposed that in oligomeric structures, the centralregion of A� monomer residues 15–21 forms a parallel �-structurewith a neighbouring A� monomer, in which the interactions areprimarily hydrophobic [13], in keeping with NMR results[11]. Ourresults suggest that A� oligomers are probably intermediate inamyloid fibril formation as some inhibitors inhibit the formationof both oligomers and fibrils. However, oligomers are not neces-sarily essential for fibrils. Oligomers may have entirely differentconformations to the �-sheet normally seen with fibrils. The preciseunderstanding of the molecular conformation involved in oligomerformation would be a fruitful line of future enquiry and enable thedesign of therapeutics that are more potent and specific towardsoligomers [3].

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