Multiple-Peptide Conjugates for Binding β-Amyloid Plaques of

86

Bioconjugate Chem. 2003, 14, 86−92

Multiple-Peptide Conjugates for Binding β-Amyloid Plaques of Alzheimer’s Disease Guobao Zhang,†,‡ Michael J. Leibowitz,§,⊥ Patrick J. Sinko,|,⊥ and Stanley Stein*,†,‡,§,|,⊥ Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, Department of Chemistry, Rutgers University, Piscataway, New Jersey 08854, Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School-UMDNJ, Piscataway, New Jersey 08854, Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, New Jersey 08854, and Cancer Institute of New Jersey, New Brunswick, New Jersey 08903. Received March 8, 2002; Revised Manuscript Received July 18, 2002

Formation of β-amyloid plaques in Alzheimer’s disease is initiated by intermolecular contact of the 5-amino acid sequence, KLVFF, in β-amyloid peptides ranging in size from 40 to 43 residues. Through optimization of binding avidity using structure/function studies, we have found that the retro-inverso peptide, ffvlk, binds artificial fibrils made from Aβ1-40 with moderate affinity (Kd ) 5 × 10-7 M). Conjugates having two copies of this peptide, whether connected by a long poly(ethylene glycol) (PEG) spacer or just two amino acids, display about 100-fold greater affinity for fibrils. Placing six copies of ffvlk on a branched PEG resulted in a 10 000-fold greater affinity (Kd ) 1 × 10-10 M) than the monomer peptide. This increased affinity was accompanied by more effective inhibition of the thioflavin T fluorescence signal, which correlates with neurotoxicity of plaques and fibrils. We propose that conjugates bearing several copies of ffvlk may be useful as diagnostic and therapeutic agents for Alzheimer’s disease.

INTRODUCTION

In Alzheimer’s disease (AD), the brain is characterized by diffuse atrophy, especially of the cortex and hippocampus, and by the presence of senile plaques, the hallmark of AD. The plaques are complex extracellular lesions composed of a central deposition of β-amyloid peptide. Genetic, neuropathological, and biochemical evidence has shown that these deposits of β-amyloid peptide play an important role in the pathogenesis of AD (Glenner et al., 1984; Master et al., 1985; Selkoe, 1994). β-Amyloid (Aβ) peptide refers to a 39-43 amino acid peptide derived from the amyloid precursor protein (APP) by proteolytic processing (Figure 1). Both the Aβ1-40and Aβ1-42 amyloid peptides are components of the deposits of amyloid fibrils found in brain tissue of AD patients. Aβ1-42 is believed to play a more important role in the early stage of fibril formation and to have a seeding effect, thus being more “amyloidogenic” than Aβ1-40. The fibril formation process is proposed to have two phases, nucleation followed by extension. The nucleation phase requires unfavorable, rate-limiting association steps from monomers. Once the nucleus of aggregated monomers has been formed, further addition of monomers becomes thermodynamically favorable, resulting in rapid extension of amyloid peptides into fibrils and then plaques (Harper and Lansbury, 1997). * Corresponding author: Stanley Stein, Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ 08854. Tel.: 732-445-3831 ext. 224. Fax: 732-445-3134. E-mail: [email protected]. † Center for Advanced Biotechnology and Medicine. ‡ Department of Chemistry, Rutgers University. § Robert Wood Johnson Medical School-UMDNJ. | Department of Pharmaceutics, Rutgers University. ⊥ Cancer Institute of New Jersey.

Figure 1. Partial sequence of APP770. The β-amyloid peptide, Aβ1-43, is shown in bold; Aβ1-40 would have the three amino acids, IAT, truncated from the C-terminus. The motif of Aβ that is the initial contact site in fibril formation is underlined.

Finding an inhibitor of the proteolytic process that is believed to excise the β-amyloid peptide from APP has been suggested as an approach to plaque elimination (Esler and Wolfe, 2001). Alternatively, plaque elimination could involve blocking or reversing the process of Aβ monomer aggregation into amyloid fibrils and plaques. This latter approach does not necessarily require a complete blockade, but is based on the hypothesis that aggregated Aβ in its disordered form is not neurotoxic. The fact that Aβ is also produced in normal people indicates that there may be certain functions associated with it (Haass et al., 1992; Seubert et al., 1992). Therefore, preventing Aβ from forming highly ordered amyloid fibrils and then plaques, rather than interfering with its synthesis and proteolytic processing, may be preferable as a therapeutic strategy. The earliest studies on the aggregation process were done by Hilbich et al. (1992). They identified the critical region of Aβ involved in amyloid fibril formation by substituting the hydrophobic amino acids in Aβ by more hydrophilic amino acids and testing the effects of these changes. Their results showed that the hydrophobic core at residues 17-20 of Aβ, LVFF, is crucial for the formation of the β-sheet structure and the amyloid properties of Aβ. The Aβ1-40 analogues, where the amino acids in 17-20 are substituted by more hydrophilic amino acids, are still able to bind to full length Aβ1-40. Further-

10.1021/bc025526i CCC: $25.00 © 2003 American Chemical Society Published on Web 12/07/2002

Multiple-Peptide Conjugates for Binding β-Amyloid Plaques

more, they were reported (Hilbich et al. 1992) to inhibit fibril formation in vitro, and therefore these analogues were suggested as therapeutic reagents for AD. Similarly, Tjernberg et al. (1996) first synthesized the 31 possible decamers (corresponding to amino acid residues 1-10 through 31-40) of the Aβ1-40 molecule on a cellulose membrane matrix. The Aβ fragments capable of binding full length Aβ were identified by radioligand binding. A series of overlapping peptides, representing a region located in the central part of Aβ (Aβ9-18 to Aβ13-22), displayed prominent binding to Aβ1-40‚Aβ11-20, which comprises the center of the binding region, was selected for further studies of the structural requirements for binding. This peptide, as well as N- and C-terminally truncated fragments, were synthesized and tested. It was found that the shortest peptide still displaying consistently high Aβ binding capacity had the sequence KLVFF (corresponding to Aβ16-20). This result agreed with Hilbich et al. (1992). This peptide was studied by microscopy and was found to be able to interfere with fibril formation in vitro. Having shown that the short peptide KLVFF can bind to Aβ and disrupt ordered fibril formation, Tjernberg et al. (1997) showed that peptide KLVFF binds stereospecifically to the homologous sequence in Aβ, i.e., Aβ16-20. Also, molecular modeling suggested that association of the two homologous sequences leads to the formation of an atypical antiparallel β-sheet structure stabilized primarily by interaction between the Lys, Leu, and Phe residues.The self-recognition property of the peptide, KLVFF, without any additional amino acids, has recently been confirmed (Watanabe et al., 2001). On the basis of these results, Ghanta et al. (1996) presented an approach to the design of inhibitors of Aβ toxicity. In their strategy, a recognition element, which interacts specifically with Aβ, is combined with a disrupting element, which alters Aβ aggregation pathways. They synthesized a peptide composed of residues 15-25 of Aβ, designed as the recognition element, linked to an oligolysine disrupting element. This inhibitor does not alter the apparent secondary structure of Aβ nor prevent its aggregation; rather, it causes changes in aggregation kinetics and higher order structural characteristics of the aggregate. In addition to its influence on the physical properties of Aβ aggregates, the inhibitor completely blocks Aβ toxicity to PC-12 cells. These results suggest that formation of disordered aggregates rather than complete blockade of amyloid fibril formation might be sufficient for abrogation of toxicity. Many peptide fragments, homologous to the β-amyloid peptide, have been synthesized and tested, and they can block the orderly aggregation of the β-amyloid peptide. For example, Soto et al. (1998) designed small peptides to interfere with the development of β-sheet structures. Their so-called ‘β-sheet breaker’, a pentapeptide with partial homology to the β-amyloid peptide, was shown to be capable of preventing β-amyloid fibril formation and disassembling preformed fibrils in vitro when a 20-fold excess of inhibitor peptide was used. However, specific binding to plaques was not shown. More recently, a peptidase-resistant congener based on the KLVFF motif, having N-methyl amino acids at alternate positions, was shown to prevent ordered fibril formation (Gordon et al., 2001). Although interesting, the ability of these β-sheet breakers to oppose the accumulation of toxic plaques has been demonstrated only in model in vitro systems. To be useful therapeutically, these inhibitory compounds must be able to cross the blood-brain barrier (BBB). Furthermore, there must be specificity in the ability of the proposed inhibitory compounds to recognize aggregates

Bioconjugate Chem., Vol. 14, No. 1, 2003 87

of β-amyoid peptide, rather than bonding and disrupting β-sheet structures in unrelated proteins. We now present results that address the issues of target specificity, in addition to β-sheet disruption. Our research focuses on the design of multifunctional conjugates. We are using branched PEG as a scaffold and the peptide motif, KLVFF, as the plaque recognition element for preparing diagnostic and therapeutic agents for AD. Studies are presented on a PEG-peptide conjugate that cannot only bind to β-amyloid plaques much more avidly than those described above, but also can efficiently disrupt ordered β-sheet formation. MATERIALS AND METHODS

Synthesis of Conjugates. Peptides were synthesized manually by solid phase, Fmoc chemistry using reagents from Bachem and other commercial sources. Note that capital letters refer to the L-isomer, whereas lower case letters refer to the D-isomer. To prepare the tandem dimer peptide, R-Fmoc (fluorenylmethoxycarbonyl), -Mtt (methoxytrityl)-Lys was placed at the C-terminus. The Fmoc group was removed by treating the resin with 20% piperidine, and Fmoc-β-alanine was coupled. After piperidine followed by 1% trifluoroacetic acid deprotection, the dimer peptide was produced by continuing the synthesis in the normal manner. Thus, the tandem dimer comprised two identical sequences, ffvlk, joined at their C-termini by a Lys-β-Ala spacer. A different dimer was produced by coupling KLVFF to R,ω-di-N-hydroxysuccinimide-PEG (Shearwater Polymers, Huntsville, AL). Peptides were cleaved from the resin and deprotected using 90% trifluoroacetic acid, 5% thioanisole, 3% ethanedithiol, and 2% anisole. After purification by reverse-phase HPLC, molecular weights were determined by MALDI-TOF mass spectrometry (Perkin-Elmer) and found to be within 1 Da of the predicted value. Unless otherwise indicated, all peptides have a free N-terminus and an amidated C-terminus. Peptide concentration was determined by absorbance at 260 nm using a standard solution of phenylalanine for calibration. The PEG conjugate of the retro-inverso peptide, ckffvlk (Scheme 1), was prepared from eight-arm-branched amino-PEG, 10 kDa total molecular weight (Shearwater Polymers). In this process, two biotin residues were appended to PEG via amide bond formation, as quantitated by the fluorescamine reaction (Udenfriend et al., 1972). This assay measures primary amines, and coupling two out of eight amino groups gives a decrease of 25% in the fluorescence signal of an aliquot compared with an aliquot of the original reaction mixture. Binding to Fibrils. The dissociation constant (Kd) for each peptide and for the PEG conjugate with preformed fibrils was determined by Scatchard plot analysis (Scatchard, 1949). This method is necessary when one of the binding components (fibrils) is a solid and cannot be expressed in units of concentration. The Aβ1-40 (Quality Control Biochemicals) was dissolved in phosphatebuffered saline (PBS) with 0.05% sodium azide to form a clear solution (0.5 mg/mL, 0.1 mM). The solution was shaken for 3 days to allow fibrils to form. The preformed fibrils were checked visually, and an aliquot of each fibril suspension was checked for β-amyloid quality by the ThT assay (see below). Fibrils are considered to be highly representative of β-amyloid plaques in the brain. The peptides and conjugates to be tested were labeled with either fluorescein isothiocyanate or [3H]acetic anhydride for quantitation. Labeled peptide that bound fibrils was separated from unbound labeled peptide by

88 Bioconjugate Chem., Vol. 14, No. 1, 2003 Scheme 1. Multipeptide Conjugate Synthesis

Zhang et al.

and emission wavelengths of 450 and 482 nm, respectively. The fluorescence was plotted against the molar ratio of test sample to Aβ1-40. RESULTS

ultrafiltration. Furthermore, the molecular weight cutoff of the ultrafilter was sufficiently high to permit even the largest nonfibrillar conjugates to pass through. In a typical binding assay, the total volume was 400 µL. In each Centricon filter unit (100 kDa cutoff), 50 µL of a preformed fibril suspension (0.5 mg/mL) and 50 µL of peptide or conjugate solutions of various concentrations, typically diluted down from 0.1 mM, were added. PBS solution was used to adjust the total volume to 400 µL. After incubation at room temperature with gentle shaking, some of the original mixture was taken, and the fluorescence or the radioactivity was measured to determine total peptide concentration. Then the mixture was centrifuged for 5 min at 5000 rpm at room temperature. The fluorescence or the radioactivity of the ultrafiltered permeate was measured to determine the free (unbound) peptide concentration. Then the ratio of bound concentration/free concentration was plotted against the bound concentration. The Kd was calculated from the slope of the linear plot. For Scatchard analysis of binding of conjugates to monomer peptide, the biotinylated conjugate was immobilized on streptavidin-coated plates. Thioflavin T Assay. The benzothiazole dye, thioflavin T (ThT), is a classical amyloid stain for senile plaques containing Aβ in AD brain. Aβ1-40 was dissolved in HPLC grade DMSO to yield a clear stock solution (10 mg/mL). Before use, the solution was centrifuged for 10 min at 12 000 rpm to remove potential fibril formed during storage. In a typical ThT assay, the test sample (peptide or peptide conjugate) was diluted serially into PBSA (PBS with 0.05% sodium azide). Then 95 µL of such diluted test sample solution was mixed with 5 µL of Aβ1-40 stock solution in a 1.5 mL polypropylene tube. The mixtures were shaken vigorously at room temperature for 2 days. Then, 500 µL of 12 µM ThT solution in sodium phosphate buffer pH 6.0 was added. The tubes were vortexed briefly. The mixture was left standing at room temperature for 30 min. Then fluorescence was measured at excitation

Binding Properties. The goal of these experiments was to maximize the avidity of interaction with amyloid plaques while preserving the natural KLVFF motif. Fibrils made similarly to our procedure are commonly used to represent amyloid plaques in the brain. According to the Scatchard equation, the data form a straight line in which the slope is the negative reciprocal of the dissociation constant. The various peptides and peptidePEG conjugates and their binding avidity to preformed fibrils are summarized in Table 1. Note the relative standard deviation of about 30% for the prototype peptide, KLVFF (Table 1), when evaluating the data. Peptide KLVFF is the natural sequence of residues 16-20 from Aβ, the nucleation site for peptide aggregation. Peptide ffvlk is a retro-inverso peptide, made of all D-amino acids and having the reverse of the natural sequence. Peptide ffvlkk is the retro-inverso peptide with one extra lysine at the C-terminus. That extra lysine provides an additional positive charge that might increase the avidity of the peptide for binding to fibrils. Peptide FLKVF is a control peptide having a scrambled sequence. We confirmed that the binding to preformed fibrils is sequence specific since the control peptide has much lower affinity than does KLVFF. We also found that the retro-inverso peptides ffvlk and ffvlkk both have slightly higher affinity (2-3-fold lower dissociation constants) with β-amyloid fibrils than does the natural KLVFF (Table 1; Figure 2A) peptide. More importantly, these retro-inverso peptides are made of all D-amino acids and are resistant to protease digestion in vivo. A PEG conjugate containing two copies of the prototype fibril-binding peptide had a dissociation constant (Kd) that was 100-fold lower than the free peptide, KLVFF. The tandem dimer peptide (Table 1), having two closely spaced copies of the retro-inverso peptide, gave a Kd of the same magnitude as the peptide-PEG-peptide conjugate having one peptide appended at either end of the polymer chain. The markedly increased avidity of both dimer peptides for preformed fibrils suggested that increasing the number of Aβ-derived peptides on a single polyvalent carrier molecule might even further increase the binding strength. Therefore, we tested the fibrilbinding ability of a hexameric conjugate of the retroinverso peptide, ckffvlk, on a branched PEG carrier. The conjugate PEG10k8a(biotin)2(ckffvlk)6 was made for this purpose. The PEG used in this conjugate was branched PEG of molecular weight 10 kDa with eight primary amines used for attaching other components. The Kd for binding of this hexameric peptide conjugate to fibrils was determined to be 1.1 × 10-10 M (Figure 2B), which is 4 orders of magnitude higher avidity than the single peptide. Thus, we have found the means to increase binding avidity by using multiple interactions between the “ligand” and the “receptor” molecules (i.e., the peptide conjugate and the fibril, respectively). Besides binding to plaques, inhibitor peptides could hypothetically interfere with plaque formation by binding to monomer or oligomeric Aβ peptides before they have a chance to form β-sheets. To test this possibility, the target was monomeric Aβ1-40 instead of fibrils. The tandem dimer peptide was found to bind Aβ1-40 moderately (Kd ) 1.7 µM), while the hexameric peptide conjugate had about 10-times greater avidity (Kd ) 0.16 µM)

Multiple-Peptide Conjugates for Binding β-Amyloid Plaques

Bioconjugate Chem., Vol. 14, No. 1, 2003 89

Table 1. Summary of Kd Values Determined by Scatchard Plot binding compound KLVFF (prototype) ffvlk (retro-inverso) ffvlkk FLKVF (scrambled) PEG-(KLVFF)2 (dimer) (ffvlk)2(βA)k (tandem dimer) PEG10k8a(biotin)2(ckffvlk)6 tandem dimer PEG10k8a(biotin)2(ckffvlk)6 ffvlk

binding target

Kd (µM)

R2 b

preformed fibrils preformed fibrils preformed fibrils preformed fibrils preformed fibrils preformed fibrils preformed fibrils fluorescein-Aβ1-40 monomer fluorescein-Aβ1-40 monomer biotinylated tandem dimer

1.1 ( 0.50 0.33 52 1.0 × 10-2 1.3 × 10-2 1.1 × 10-4 1.7 0.16 1.8

0.998 0.973 0.985 0.997 0.917 0.910 0.980 0.980 0.862 0.973

0.3a

a Standard deviation for three analyses done on different days. Scatchard plot was done on triplicate samples at each concentration in one experiment for each of the other binding compounds. b Correlation coefficient.

Figure 3. Specificity of binding. Binding of [3H]-PEG(ckffvlk)6(biotin)2 to preformed fibrils was done in the presence and absence of bovine serum albumin. The percent nonspecific binding to albumin was essentially independent of concentration of conjugate. Triplicate samples were analyzed at each concentration within the same experiment.

Figure 2. Scatchard plot analysis of binding to preformed fibrils. Panels A and B are for the peptide, KLVFF, and the conjugate PEG(ckffvlk)6(biotin)2, respectively. Both peptides have a free N-terminus and an amidated C-terminus. The peptides were labeled using tritiated acetic anhydride. Triplicate samples were analyzed at each concentration within the same experiment.

for the Aβ1-40 peptide (Table 1). By having six copies of the binding peptide present in each conjugate, there is a 10-fold greater binding (relative to the dimeric peptide) when the target is monomer peptide. In contrast, when the target is fibrils, there is a 100-fold difference between the dimeric and hexameric binding peptides. However, binding constants alone are not predictors of in vivo antiplaque activity. Specificity of binding was tested by measuring the binding to preformed fibrils in the presence of a large excess of serum albumin. That is, if some form of the KLVFF peptide were to be used clinically, would albumin interfere with binding to fibrils? Not only is albumin the most abundant protein in blood, but it is known to bind hydrophobic substances, such as the anticancer drug,

camptothecin (Mi et al., 1995). As seen in Figure 3, there was about a 60% decrease in binding of the six-peptide conjugate across the entire dosage range in the presence of bovine serum albumin at 10 mg/mL. In this assay, the amount of Aβ1-40 peptide was only 0.017 mg/mL, about 600-fold lower than albumin. Indeed, the amount of sixpeptide conjugate tested was as low as 0.01 nM (10-14 mol/mL), which is on the order of 0.01 ng/mL or 1 part per trillion of albumin. Thus, the pharmacokinetic result of this interaction might be to extend the in vivo halflife of the KLVFF-related peptides and conjugates. Albumin does not appear to sequester the binding peptide conjugate in an inactive form. Inhibition of Ordered Fibril Formation. While the Scatchard plot is a good way to evaluate a peptide or conjugate by determining the Kd for binding to preformed fibrils, it does not provide information on inhibition of ordered fibril formation, as opposed to disordered aggregation. ThT binds rapidly and specifically to the antiparallel β-sheet fibrils formed from synthetic Aβ1-40, but it does not bind to monomer or oligomeric intermediates (LeVine, 1993). Aβ in the β-amyloid fibrils in senile plaque is arranged in a highly ordered, condensed antiparallel β-sheet structure, and neuronal toxicity is attributed to this structural characteristic. That is, an amorphous aggregate of amyloid peptide is not neurotoxic, while a highly ordered, condensed antiparallel β-sheet structure is neurotoxic. For this reason, completely preventing the aggregation of Aβ may not be necessary for achieving a therapeutic effect. It has been shown that the fluorescence in the ThT assay is correlated with both neurotoxicity and with the aggregation morphology being highly ordered (Ghanta et al., 1996).

90 Bioconjugate Chem., Vol. 14, No. 1, 2003

Zhang et al.

Figure 5. Kinetics of fibril formation. A fresh solution of Aβ1-40 (0.1 mM), spiked with fluorescein-labeled Aβ1-40, was incubated with or without an inhibitor peptide (0.5 mM). Samples were centrifuged at either 2 or 24 h, and peptide content in the pellet (if present) and supernatant were measured by fluorescence. Results are the average data from two separate experiments.

Figure 4. Thioflavin T assays for inhibition of ordered fibril formation. The monomer peptide, Aβ1-40, was incubated with each test sample for 2 days to determine inhibition of ordered fibril formation. Amorphous aggregates are observed as low fluorescence intensity, while well-ordered fibrils are observed as high fluorescence signal. Each of panels A, B, and C depict the concentration curves for several variations in the KLVFF motif done at the same time. Triplicate samples were analyzed at each concentration within the same experiment.

The inhibitory effect of the various peptides and conjugates in preventing ordered fibril formation, as monitored by the ThT assay, is presented in Figure 4. While peptide KLVFF and retro-inverso ffvlkk show an inhibitory effect in reducing the fluorescence signal, corresponding to preventing ordered fibril formation, inverso peptides klvff and klvffk were found to be relatively ineffective at all concentrations (Figure 4A). This result confirmed that the effect on ordered aggregation of Aβ1-40 by KLVFF analogues is specific with respect to both sequence and three-dimensional structure. The

effect on ordered aggregation of Aβ1-40 by the positive charge at the N-terminus of the retro-inverso peptide, ffvlk, was examined using the N-acetylated peptide, N-acetyl-ffvlkk; there was no suppression of ThT fluorescence even at a 6-fold excess of the hexapeptide. However, an extra lysine residue in the peptide, N-acetylkffvlkk, which restored a positive charge at the Nterminus, was able to inhibit the ThT fluorescence identically to the retro-inverso hexapeptide, ffvlkk (Figure 4B). Indeed, there was actually an increase in ThT fluorescence with N-acetyl-ffvlkk. In a repeat experiment with N-acetyl-ffvlkk, this increase was qualitatively reproducible, including the large variability in the fluorescence signal (Figure 4B). The tandem dimer retro-inverso peptide was found to be much more effective in preventing ordered fibril formation than the prototype peptide, KLVFF, or any of the monomer peptides, as indicated by both the amount of fluorescence reduction in the ThT assay and the relative amount needed for inhibition (Figure 4C). The conjugate PEG10k8a(biotin)2(peptide)6 was even more effective in preventing the ThT signal (Figure 4C). Indeed, the six-copy conjugate is about 3 times more potent than the dimer when expressed per mole of peptide subunit, as in Figure 4C, but 9 times more potent when expressed per mole of conjugate molecule. That is, maximal inhibition is attained at a ratio of one inhibitor peptide per three Aβ1-40 peptides in the context of the conjugate, but at a 1:1 ratio in the tandem dimer form of the same retroinverso peptide. Other Studies. Experiments were done to determine if the peptides or conjugates can disaggregate preformed fibrils. Preformed fibrils were incubated with peptides or conjugates at different ratios for 3 days at room temperature. Then the ThT assay was done to quantify the amount of ordered fibrils remaining after treatment. None of the peptides or peptide conjugates were able to break preformed fibrils under these conditions, according to the ThT assay (data not shown). The kinetics of aggregation of Aβ1-40 was also monitored in the absence and presence of inhibitory peptides. Aβ1-40 was incubated under conditions typically used to prepare fibrils, normally taking 2-3 days to complete. Since the transition from a soluble to an insoluble form of Aβ1-40 was at about 15 h (data not shown), the experimental samples were spun down at either 2 or 24 h to observe any change in the kinetics of aggregation. As shown in Figure 5, all of the Aβ1-40 peptide was in

Multiple-Peptide Conjugates for Binding β-Amyloid Plaques

the supernatant at 2 h, while all was in the pellet at 24 h, in agreement with the transition occurring at 15 h. However, in the presence of either the dimer or the sixcopy conjugate inhibitor, all of the Aβ1-40 appeared in the pellet after only 2 h. On the basis of the ThT assay (Figure 4C), this increased kinetics results in nonordered aggregates rather than highly ordered beta-sheet structures, in agreement with Ghanta et al. (1996). Essentially the same conclusion, that a decrease in the rate of aggregation (i.e., transition time going from 30 to 43 h) of Aβ increased the tendency to form β-amyloid plaques, was drawn in studies on the Flemish variant, Ala692Gly, which results in early onset AD (Walsh et al., 2001). DISCUSSION

This work confirms and extends the structure/function studies on Aβ fibrils of the peptide motif, KLVFF. We show that the retro-inverso analogue, ffvlk, is preferable for use as a target recognition element. Not only does it have a 2-fold greater affinity for fibrils, but it is composed of peptidase-resistant D-amino acids. According to the ThT assay, a positively charged group at the N-terminus is essential in order to inhibit ordered fibril formation. Furthermore, this work demonstrates the cooperative effect that multiple copies of the KLVFF motif can have on the binding to fibrils and the aggregation of Aβ1-40 into fibrils. Two different dimers displayed 100-fold stronger avidity than their respective monomers for fibrils. In one dimeric form, (KLVFF)-PEG-(KLVFF), the two copies of the L-amino acid binding peptide were linked by a long, flexible PEG chain. In the other dimeric form, the two copies of the retro-inverso peptide, fflvlk, were linked by just a pair of amino acids. The synergistic effect is so substantial that a six-copy conjugate of the retro-inverso peptide, kffvlk, can bind preformed fibrils 10 000-times more avidly than does the monomer peptide. Furthermore, the multicopy conjugates block the formation of well ordered fibrils from monomer Aβ peptide. Thus, the KLVFF motif might be useful not only as a target recognition element, but also as a β-sheet breaker. The concept of increased avidity due to multivalent interactions is certainly not new. For example, combining multiple weak but specific receptor-ligand interactions was recently illustrated by Mourez et al. (2001). Using phage display libraries directed to the heptameric cellbinding subunit of anthrax toxin, they selected a peptide sequence, synthesized that peptide, and appended multiple copies to a flexible polymer. This polyvalent inhibitor was able to block toxin action in an animal model. Similarly, we showed the relationship between the number of copies of the macrophage chemoattractant peptide, N-formyl-Met-Leu-Phe-OH, and macrophage receptor avidity, being 1000-fold stronger with the eightcopy versus the single-copy PEG adducts (Pooyan et al., 2002). In this study, the biotin groups were included in the hexameric conjugate for future use as a reporter for a quantitative ELISA or for a qualitative tissue stain. Recently, we have described the ability of an appended biotin group to impart oral bioavailability to a fragment of the HIV-1-encoded Tat protein (Ramanathan et al. 2001a). The permeability across the intestinal barrier was even greater for PEG conjugates comprising as many as eight copies of the Tat peptide (Ramanthan et al. 2001b). Indeed, the membrane-penetrating properties of Tat peptide and other small cationic peptides has been documented by several laboratories, as reviewed by

Bioconjugate Chem., Vol. 14, No. 1, 2003 91

Fischer et al. (2001). Since delivery of plaque-binding compounds across the blood-brain barrier (BBB) might be a requirement for a diagnostic or therapeutic agent for AD, the presence of biotin or another transport ligand might be important for clinical applications. However, recent results (DeMattos et al., 2002) suggest that a therapeutic or diagnostic agent, comprising a monoclonal antibody against Aβ peptide in their studies, can be useful when administered peripherally. Apparently, the pools of soluble plaque-forming Aβ peptide in the central nervous system and in the periphery are in equilibrium, implying that Aβ is able to cross the BBB. Injection of their antibody was shown to produce a “sink” effect, drawing soluble Aβ from the brain into the bloodstream (DeMattos et al., 2002). This could remove Aβ from the brain, potentially halting or even reversing plaque formation. The antibody results suggest that even if the multivalent conjugates described above do not cross the BBB, they may be useful for diagnosis and treatment of AD. LITERATURE CITED (1) DeMattos, R B., Bales, K. R., Cummins, D. J., Paul, S. M., and Holtzman, D. M. (2002) Brain to plasma β-amyloid efflux: a measure of brain amyloid burden in a mouse model of Alzheimer’s disease. Science 295, 2264-2267. (2) Esler, W. P., and Wolfe, M. S. (2001) A portrait of Alzheimer secretases-New features and familiar faces. Science 293, 1449-1454. (3) Fischer, P. M., Krausz, E., and Lane, D. P. (2001) Cellular delivery of impermeable effector molecules in the form of conjugates with peptides capable of mediating membrane translocation. Bioconjugate Chem. 12, 825-841. (4) Ghanta, J., Shen, C. L, Kiessling, L. L., and Murphy, R. M. (1996) A strategy for designing inhibitor of β-Amyloid toxicity, J. Biol. Chem. 271, 29525-29528. (5) Glenner, G., and Wong, C. W. (1984) Alzheimer’s Disease: Initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem. Biophys. Res. Commun. 120, 885-890. (6) Gordon, D. J., Sciarretta, K. L., and Meredith, S. C. (2001) Inhibition of β-amyloid(40) fibrillogenesis and disassembly of β-amyloid(40) fibrils by short β-amyloid congeners containing N-methyl amino acids at alternate residues. Biochemistry 40, 8237-8245. (7) Haass, C., Schlossmacher, M. G., Hung, A. Y., Vigo-Pelfrey, C., Mellon, A., Ostaszewski, B. L., Lieberburg, I., Koo, E. H., Schenk, D., and Teplow, D. B. (1992) Amyloid beta-peptide is produced by cultured cells during normal metabolism. Nature 359, 322-325. (8) Harper, J. D., and Lansbury, P. T. (1997) Models of amyloid seeding in Alzheimer’s disease and scrapie: Mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins. Annu. Rev. Biochem. 66, 385407. (9) Hilbich, C., Kisters-Woike, B., Reed, J., Masters, C. L., and Beyreuther, K. (1992) Substitutions of hydrophobic amino acids reduce the amyloidogenicity of Alzheimer’s disease βA4 peptides. J. Mol. Biol. 228, 460-473. (10) LeVine, H. (1993), Thioflavin T interaction with synthetic Alzheimer’s disease β-amyloid peptides: Detection of amyloid aggregation in solution. Protein Sci. 2, 404-410. (11) Master, C. L., Simms, G., Weinman, N. A., Multhap, G., Mcdonald, B. L., and Beyreuther, K. (1985) Amyloid plaque core protein in Alzheimer Disease and Down Syndrome. Proc. Natl. Acad. Sci. U.S.A. 82, 4245-4249. (12) Mi, Z., Malak, H., and Burke, T. G. (1995) Reduced albumin binding promotes the stability and activity of topotecan in human blood. Biochemistry 34, 13722-13728. (13) Mourez, M., Kane, R. S., Mogridge, J., Metallo, S., Deschatelets, O., Sellman, B. R., Whitesides, G. M., and Collier, R. J. (2001) Designing a polyvalent inhibitor of anthrax toxin. Nature Biotechnol. 19, 958-961.

92 Bioconjugate Chem., Vol. 14, No. 1, 2003 (14) Pooyan, S., Qiu, B., Chen, M., Fong, D., Sinko, P., Leibowitz, M. J., and Stein, S. Conjugates bearing multiple formylmethionyl peptides display enhanced binding to but not activation of phagocytic cells. Bioconjugate Chem. 13, 216223. (15) Ramanathan, S., Pooyan, S., Stein, S., Prasad, P. D., Wang, J., Leibowitz, M. J., Ganapathy, V., and Sinko, P. (2001a) Targeting the sodium dependent multivitamin transporter (SMVT) for improving the oral absorption properties of a retro-inverso Tat nonapeptide. Pharm. Res. 18, 950-956. (16) Ramanathan, S., Pooyan, S., Qiu, B., Zhang, G., Stein, S., Leibowitz, J., and Sinko, P. (2001b) Targeted PEG-based bioconjugates enhance the cellular uptake and transport of an HIV-1 Tat nonapeptide. J. Controlled Release 77, 199212. (17) Scatchard, G. (1949) The attraction of proteins for small molecules and ions. Ann. New York Acad. Sci. 51, 660-672. (18) Selkoe, D. J. (1994) Cell biology of the amyloid-protein precursor and the mechanism of Alzheimer’s disease. Annu. Rev. Cell Biol. 10, 373-403. (19) Seubert, P., Vigo-Pelfrey, C., Esch, F., Lee, M., Dovey, H., Davis, D., Sinha, S., Schlossmacher, M., Whaley, J., and Swindlehurst, C. (1992) Isolation and quantification of soluble Alzheimer’s β-peptide from biological fluids. Nature 359, 325327. (20) Soto, C., Siguardsson, E. M., Morelli, L., Kumar, R. A., Castan˜o, E. M., and Frangione, B. (1998) β-sheet breaker

Zhang et al. peptides inhibit fibrillogenesis in a rat brain model of amyloidosis: Implications for Alzheimer’s therapy. Nat. Med. 4, 822-826. (21) Tjernberg, L. O., Lilliehook, C., Callaway, D. J., Na¨slund, J., Hahne, S., Thyberg, J., Terenius, L., and Nordstedt, C. (1997) Controlling amyloid β-peptide fibril formation with protease-stable ligands. J. Biol. Chem. 272, 12601-12605. (22) Tjernberg, L. O., Naslund, J., Lindqvist, F., Johansson, J., Karlstro¨m, A. R., Thyberg, J., Terenius, L., and Nordstedt, C. (1996) Arrest of β-amyloid fibril formation by a pentapeptide ligand. J. Biol. Chem. 271, 8545-8548. (23) Udenfriend, S., Stein S., Bohlen P., and Dairman, W. (1972) Applications of fluorescamine, a new reagent for assay of amino acids, peptides, proteins and other primary amines in the picomole range. Science 178, 871-872. (24) Walsh, D. M., Hartley, D. M., Condron, M. M., Selkoe, D. J., and Teplow, D. B. (2001) In vitro studies of amyloid protein fibril assembly and toxicity provide clues to the aetiology of Flemish variant (Gly692Ala) Alzheimer’s disease. Biochem. J. 355, 869-877. (25) Watanabe, K., Segawa, T., Nakamura, K., Kodaka, M., Konakara, T., and Okuno, H. (2001) Identification of the molecular interaction site of amyloid β peptide by using a fluorescence assay. J. Pept. Res. 58, 342-346.

BC025526I