Biochemistry 2010, 49, 3261–3272 3261 DOI: 10.1021/bi100144m
Development of a Proteolytically Stable Retro-Inverso Peptide Inhibitor of β-Amyloid Oligomerization as a Potential Novel Treatment for Alzheimer’s Disease† )
Mark Taylor,‡ Susan Moore,‡ Jennifer Mayes,‡ Edward Parkin,‡ Marten Beeg,§ Mara Canovi,§ Marco Gobbi,§ David M. A. Mann, and David Allsop*,‡ ‡
Division of Biomedical and Life Sciences, School of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, U.K., Department of Biochemistry and Molecular Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, Milano, Italy, and Clinical Neurosciences Research Group, University of Manchester, Hope Hospital, Salford M6 8HD, U.K. )
§
Received January 29, 2010; Revised Manuscript Received March 15, 2010
The formation of β-amyloid (Aβ) deposits in the brain is likely to be a seminal step in the development of Alzheimer’s disease. Recent studies support the hypothesis that Aβ soluble oligomers are toxic to cells and have potent effects on memory and learning. Inhibiting the early stages of Aβ aggregation could, therefore, provide a novel approach to treating the underlying cause of AD. We have designed a retroinverso peptide (RI-OR2, H2N-rrGrkrlrvrfrfrGrr-Ac), based on a previously described inhibitor of Aβ oligomer formation (OR2, H2N-R-G-K-L-V-F-F-G-R-NH2). Unlike OR2, RI-OR2 was highly stable to proteolysis and completely resisted breakdown in human plasma and brain extracts. RI-OR2 blocked the formation of Aβ oligomers and fibrils from extensively deseeded preparations of Aβ(1-40) and Aβ(1-42), as assessed by thioflavin T binding, an immunoassay method for Aβ oligomers, SDS-PAGE separation of stable oligomers, and atomic force microscopy, and was more effective against Aβ(1-42) than Aβ(1-40). In surface plasmon resonance experiments, RI-OR2 was shown to bind to immobilized Aβ(1-42) monomers and fibrils, with an apparent Kd of 9-12 μM, and also acted as an inhibitor of Aβ(1-42) fibril extension. In two different cell toxicity assays, RI-OR2 significantly reversed the toxicity of Aβ(1-42) toward cultured SH-SY5Y neuroblastoma cells. Thus, RI-OR2 represents a strong candidate for further development as a novel treatment for Alzheimer’s disease. ABSTRACT:
There is substantial evidence from molecular genetics, transgenic animal studies, and aggregation/toxicity studies to suggest that the conversion of the β-amyloid (Aβ)1 peptide from soluble monomers to aggregated forms in the brain is a key event in the pathogenesis of Alzheimer’s disease (AD). It seems increasingly likely that early “soluble oligomers” are responsible for the reported toxic effects of Aβ, rather than fully formed amyloid fibres, and these small oligomers could be one of the causes of neurodegeneration in vivo (1-9). They have also been reported to have potent effects on synaptic plasticity, learning, and memory in animal models (2, 3, 7). Inhibition of toxic Aβ oligomer formation is therefore a potential therapeutic target for AD (10). Considerable progress has already been made in discovering inhibitors of Aβ aggregation and toxicity (11, 12). One of the strategies employed has been the use of peptide-based inhibitors (13). † This research was supported by The Alzheimer’s Research Trust. *To whom correspondence should be addressed: Division of Biomedical and Life Sciences, School of Health and Medicine, Lancaster University, Lancaster LA1 4YQ, U.K. Telephone: 44-1524-592122. Fax: 44-1524-593192. E-mail:
[email protected]. 1 Abbreviations: Aβ, β-amyloid peptide; AD, Alzheimer’s disease; AFM, atomic force microscopy; BSA, bovine serum albumin; ByBOP, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; ECL, enhanced chemiluminescence; HFIP, hexafluoro-2-propanol; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; LDH, lactate dehydrogenase; MTS, 3-(4,5-dimethylthiazol-2yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PB, phosphate buffer; PBS, phosphate-buffered saline; SDS-PAGE, polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulfate; SPR, surface plasmon resonance; TFA, trifluoroacetic acid; Th-T, thioflavin T.
These have been focused generally on the internal Aβ(16-22) sequence, KLVFFAE, because this region has been reported to be primarily responsible for the self-association and aggregation of the peptide (14, 15). Soto and co-workers have designed “β-sheet breaker peptides” by incorporating proline residues into this peptide sequence (16, 17). Another strategy has used N-methylated peptides (18, 19), which act by binding to Aβ through one hydrogenbonding face while blocking the propagation of the hydrogen bond array of the β-sheet with the other non-hydrogen-bonding face. Murphy and co-workers (20) have reported a different strategy, based on residues 15-25 of Aβ linked to an oligolysine disrupting element. However, these latter inhibitors appear not to prevent Aβ aggregation but cause a change in aggregation kinetics and higherorder structural characteristics of the aggregate (20). Some further modifications of these peptide inhibitors have included the incorporation of solubilizing (charged) residues (21), the use of D-amino acids to improve stability (21, 22), multivalent peptides for increased potency (23), and the attachment of targeting sequences to enhance cell and blood-brain barrier permeability (21). It should be noted that these past studies have generally utilized techniques such as turbidity, thioflavin-T binding, sedimentation, and Congo red binding to test for inhibitors of Aβ aggregation. These methods can identify only compounds that are capable of inhibiting the formation of Aβ fibrils, so their effects on toxic oligomer formation are often not clearly established. A peptide-based inhibitor, named OR2, with the amino acid sequence RGKLVFFGR-NH2, was shown previously to inhibit the formation of early oligomeric forms of Aβ (24). This peptide, with an amidated C-terminus, was a better inhibitor of Aβ
r 2010 American Chemical Society
Published on Web 03/15/2010
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FIGURE 1: Structures of OR1, OR2, and RI-OR2 (retro-inverso OR2). L-Amino acids are in uppercase and D-amino acids in lowercase, with the direction of peptide bonds indicated by arrows. There are no separate enantiomers of glycine, which is represented in uppercase.
oligomer formation and associated Aβ toxicity than the same peptide with a conventional C-terminus [called OR1 (see Figure 1)] (24). These OR1 and OR2 peptides were designed from the central region of Aβ (KLVFF, residues 16-20), which, as noted above, is part of the binding region responsible for its self-association (14, 15). However, to aid the solubility of these peptide inhibitors, and at the same time to prevent them from self-aggregating and so acting as a “seed” to promote Aβ aggregation, an additional cationic Arg was added at their N- and C-termini, in each case via Gly as a spacer (24). The intention of placing these Gly residues as spacers between Arg and the KLVFF binding sequence was to facilitate the interaction between the inhibitor peptides and native Aβ (24). A similar strategy has been employed for the development of successful inhibitors of R-synuclein aggregation, as a potential novel treatment for Parkinson’s disease and related disorders (25). Although OR2 was shown to be an effective inhibitor of the formation of Aβ oligomers (24), there are many potential sites for proteolytic attack on this peptide, so it is unlikely itself to be a viable drug candidate. To attempt to avoid this problem but retain the antiaggregational properties of this peptide, we have now designed a “retro-inverso” version (26, 27) of OR2 (designated RI-OR2). This is a more sophisticated approach than simply replacing some or all of the L-amino acids with D-amino acids (21, 22). In a retro-inverso peptide, all of the natural L-amino acids are replaced with the D-enantiomer, but the peptide bonds are also reversed (26, 27). In theory, this should maintain a threedimensional shape similar to that of the “parent” peptide molecule and so help to retain biological activity. Here we report the results of Aβ aggregation, binding, and toxicity studies with this new RI-OR2 peptide inhibitor, as well as studies of the stability of the peptide inhibitor to proteolytic degradation. EXPERIMENTAL PROCEDURES Aβ Peptides. Recombinant Aβ(1-40) and Aβ(1-42) peptides (Ultrapure) were used for all of the experiments described below, except for the binding experiments involving the surface plasmon resonance (SPR) technique. The recombinant peptides were purchased from rPeptide Co. (Bogart, GA) and were already treated with hexafluoro-2-propanol (HFIP). Unless indicated otherwise, prior to use, the peptides were twice deseeded again in HFIP and dried using centrifugal evaporation before being dissolved in 10 mM phosphate buffer (PB) (pH 7.4) and sonicated for 4 30 s at 12 μm amplitude using an MSE sonicator. For the SPR experiments only, Aβ(1-42) was synthesized first of all as the depsi-peptide molecule described by Taniguchi et al. (28). The depsi-peptide is much more soluble than the native peptide, thus avoiding the need of vortexing for its dissolution, and also has a much lower propensity to aggregate, thereby preventing the spontaneous formation of seeds in solution. The native Aβ(1-42) peptide was then obtained from the depsipeptide by a “switching” procedure involving a change in
Taylor et al. pH (28). The Aβ(1-42) peptide solution obtained immediately after switching is seed-free as shown in carefully conducted previous work (28, 29). To prepare Aβ(1-42) fibrils, the switched peptide solution was diluted with water to 100 μM, acidified to pH 2.0 with 1 M HCl, and left to incubate for 24 h at 37 °C. The presence of amyloid fibrils was confirmed by atomic force microscopy (AFM) (29) (see also Figure S2C of the Supporting Information). Peptide Inhibitors. Inhibitory peptides OR2 and RI-OR2 were made by Cambridge Peptides (Birmingham, U.K.). OR2 was made on a conventional automated amino acid synthesizer, whereas RI-OR2 was made by manual Fmoc synthesis, using (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (ByBOP) as a coupling agent, on Rink amide MBHA resin. After purification by reverse-phase high-performance liquid chromatography (HPLC), the peptides were >95% pure. Stability of Inhibitors to Proteolysis. The stability of the inhibitors in plasma and brain extracts was assessed by addition of 10 μL of filtered (0.22 μm filter) human blood plasma (50 mg/ mL) or postmortem human brain tissue extract (5 mg/mL) to 100 μL of filtered inhibitor (1 mM) in PBS. The brain tissue extract was prepared by macerating a small (∼50 μg) piece of frozen brain frontal cortex in 100 μL of sodium acetate buffer (pH 7.4). The inhibitor, in plasma or brain extract, was incubated at 37 °C for 24 h, loaded onto a Jupiter C18 reversed-phase HPLC column (Phenomenex) attached to a Dionex HPLC system, and separated using a gradient from 0 to 60% acetonitrile, containing 0.1% trifluoroacetic acid, over 30 min. Analysis of peptide recovery was performed with Chromeleon software. The absorbance was measured at 220 nm. The stability of inhibitors to individual proteases was assessed following incubation of a 1 mM solution of inhibitor and 0.1 mg/mL purified protease for 24 h at 37 °C in 10 mM PB (pH 7.4) (except in the case of pepsin, where the pH was lowered to 4). Each solution (30 μL) was loaded onto a Jupiter C18 reversed-phase HPLC column, and the peptide was eluted and analyzed as described above. Thioflavin T Assays. Th-T assays were conducted in 96-well clear-bottom microtiter plates (NUNC) by incubating the Aβ peptides (20 μM) at 25 °C in the continuous presence of Th-T (10 μM) in 10 mM PB (pH 7.4). Inhibitor, when present, was at molar ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 relative to Aβ, and the total volume of the solution in each well was 100 μL. The plates were shaken and read every 10 min (λex = 442 nm, and λem = 483 nm) in a BioTek Synergy plate reader. Immunoassay for Oligomeric Aβ. This sandwich immunoassay follows the method described previously by Moore et al. (30). Briefly, 96-well ELISA plates (Maxisorb) were coated with mouse monoclonal antibody 6E10 diluted 1:1000 in assay buffer [Tris-buffered saline (TBS) (pH 7.4) containing 0.05% γ-globulins and 0.005% Tween 20]. The incubated samples of peptide, with or without inhibitor (20 μM Aβ in 10 mM PB and inhibitor:Aβ molar ratios of 2:1, 1:1, and 1:2), were diluted to 1 μM Aβ and incubated, in triplicate, in the 96-well plates for 1 h at 37 °C. The plates were washed with 10 mM phosphatebuffered saline (PBS), containing 0.5% Tween 20 (PBS-T). Following this, 100 μL of TBS containing a 1:1000 dilution of biotinylated 6E10 was added to each of the 96 wells and incubated for 1 h at 37 °C. After the samples had been washed further, europium-linked streptavidin was added at 1:500 dilution in StrepE buffer (TBS containing 20 μM DTPA, 0.5% bovine serum albumin, and 0.05% γ-globulins), again incubated
Article for 1 h, and washed. Enhancer solution was added, and the plates were read using the time-resolved fluorescence setting for europium on a Wallac Victor 2 plate reader. Detection of Oligomers by SDS-PAGE and Immunoblotting. For these experiments, Aβ(1-42) was deseeded to a monomeric form, based on a modification of the method described by Manzoni et al. (31). The peptide (1 mg) was dissolved in 1 mL of trifluoroacetic acid (TFA) containing 45 μL of thioanisole in a glass container. This was incubated for 1 h at room temperature and sonicated (4 30 s) in an ice-water bath every 15 min. The liquid was evaporated using a stream of nitrogen gas, and the peptide was redissolved in 1 mL of HFIP. This was sonicated (4 30 s in an ice-water bath) after incubation for 10 min at room temperature. The HFIP was removed by centrifugal evaporation and the step repeated. Following this, the protein was again dissolved in HFIP, sonicated, and then centrifuged at 15700g at room temperature to pellet any aggregated peptide. The liquid was decanted before being divided into convenient aliquots and the HFIP removed by centrifugal evaporation. Dried peptide was stored at -20 °C. To assess the ability of RI-OR2 to inhibit the formation of small oligomers of Aβ(1-42), we followed a slightly modified method of Chafekar et al. (23). Deseeded Aβ(1-42) was dissolved at a concentration of 5 μM in 10 mM PB (pH 7.4) containing 0, 5, or 25 μM RI-OR2. A sample was taken (time zero), and then the solutions were incubated at 37 °C for 4 h. Each solution was mixed with Novex tricine SDS sample buffer (Invitrogen) and loaded, without boiling, onto a Novex 10-20% tricine gel (Invitrogen). The gel was run for 1 h at 100 V and then protein transferred by Western blot onto a nitrocellulose membrane. The membrane was probed using mouse monoclonal antibody 6E10 at 1:10000 in 5% dried skimmed milk powder in PBS with 0.5% Tween. After any unbound antibody had been removed by washing with PBS Tween, the membrane was probed with a 1:5000 dilution of HRP-linked rabbit anti-mouse antibody (Sigma) in 5% milk powder as described above. Following another washing step, as before, bands were visualized using the Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate and detected using Amersham Hyperfilm ECL. Atomic Force Microscopy. Atomic force microscopy (AFM) was conducted on a Digital Instruments Multimode Scanning Probe Microscope using tapping mode. Aβ(1-42), preincubated for 24 h at 25 °C in 10 mM PB (pH 7.4) with and without inhibitor (at a 1:1 molar ratio), was spotted onto freshly cleaved mica sheets and allowed to dry. Three scans were performed for each condition. Cell Toxicity Studies. SHSY-5Y cells were maintained in Ham’s F12 and EMEM medium mixed at a 1:1 ratio containing 2 mM glutamine, 1% nonessential amino acids, and 15% fetal calf serum supplemented with a 500 μg/mL penicillin/streptomycin solution. Cells were transferred to a sterile 96-well plate with approximately 25000 cells per well and allowed to acclimatize for 48 h. The Ham’s F12/EMEM medium was removed by suction and replaced with Optimem medium (100 μL/well) containing either no Aβ or Aβ(1-42) (10 μM, preincubated on its own for 24 h in PB), with and without the OR2 and RI-OR2 inhibitors (10 μM). The cells were left for 24 h and then assessed. The toxicity of the inhibitors was assessed using the CellTiter 96 Aqueous One Solution Cell Proliferation (MTS) Assay (Promega) and CytoTox-ONE Homogeneous Membrane Integrity (LDH) Assay (Promega). Surface Plasmon Resonance Experiments. These experiments were conducted using a Bio-Rad ProteOn XPR36 SPR
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machine (32). Aβ(1-42) monomers or fibrils (see Aβ peptides), with bovine serum albumin (BSA) as a control, were immobilized in parallel-flow channels of the same GLC sensor chip (Biorad) using amine coupling chemistry. Briefly, after surface activation, the monomeric or fibrillar peptide preparations were diluted to 10 μM in acetate buffer (pH 4.0) and then injected for 5 min at a flow rate of 30 μL/min. Any remaining activated groups were blocked with ethanolamine (pH 8.0). The final immobilization levels were similar in each case, with both being ∼2500 resonance units (1 RU = 1 pg of protein/mm2). An empty “reference” surface was prepared in parallel using the same immobilization procedure, but without addition of the peptide. Both Aβ(1-42) monomers and fibrils immobilized on the sensor chip bound 6E10 antibody, whereas fibrils bound only Congo red (M. Gobbi, personal communication). Sensorgrams were then obtained via injection of three different concentrations of RI-OR2 (3, 10, and 30 μM), as well as the vehicle (PBS with 0.005% Tween 20), over the immobilized ligands or control surfaces, in parallel, at the same time. In a second set of experiments, further sensorgrams were obtained via injection of Aβ(1-42) monomers (3 μM), RIOR2 alone (30 μM), or a combination of the two (preincubated for 15 min before injection) into the flow chambers. RESULTS Stability of Inhibitors to Proteolytic Degradation. The stability of the inhibitors to protease action was assessed by incubating them in the presence of either a human blood plasma or brain extract, followed by HPLC analysis to quantify the amount of intact peptide remaining in solution. After an incubation period of 24 h, OR2 was almost completely degraded by either the human plasma or the brain extract, whereas the amount of RI-OR2 remained at (or close to) 100% (Figure 2A,B). Studies with individual proteolytic enzymes showed that OR2 was sensitive to degradation by elastase, cathepsin G, kallikrein, thrombin, trypsin, and chymotrypsin. In contrast, RI-OR2 was completely resistant to attack by these proteases (Figure 2C). Thioflavin T Assay of Inhibition of Aggregation. The aggregation of Aβ(1-40) and Aβ(1-42) into Th-T positive amyloid fibrils was first of all monitored by incubation of each peptide (20 μM) in the continuous presence of Th-T (10 μM) in a 96-well microtiter plate, with shaking and then measurement of fluorescence every 10 min, for periods of up to 48 h. Panels A and B of Figure 3 show data for the first 22 h, for each peptide. Aβ(1-40) exhibited a lag time (corresponding to the nucleation phase) of ∼16 h, before a period of rapid fibril formation, which was complete after 18-20 h, whereas Aβ(1-42) exhibited virtually no lag period and achieved maximal fluorescence after only 2-3 h. RI-OR2, at an equimolar concentration to Aβ, almost completely eliminated the development of Th-T fluorescence over these time periods, for both Aβ(1-40) and Aβ(1-42). The data presented in Figure 3A,B are from a single experiment but are representative of three separate experiments. After 22 h in the presence of OR2, approximately 40% of control Th-T fluorescence (after subtraction of background) was reached for Aβ(1-40), compared to
