Pharmacological Profiles of Peptide Drug Candidates for the Treatment of Alzheimer's Disease*

Céline AdessiDagger , Marie-José FrossardDagger , Christophe Boissard§, Santiago FragaDagger , Sylvain BielerDagger , Thomas RuckleDagger , Francis VilboisDagger , Sandra M. Robinson, Manfred Mutter§, William A. Banks, and Claudio SotoDagger ||

From the Dagger  Serono Pharmaceutical Research Institute, 1228 Geneva, Switzerland, § Ecole Politechnique Federal de Lausanne, 1015 Lausanne, Switzerland, and  Geriatrics Research, Educational, and Clinical Centers, Veterans Affairs Medical Center, Saint Louis University School of Medicine, St. Louis, Missouri 63106

Received for publication, November 25, 2002, and in revised form, February 3, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Amyloid plaques in brain, composed of aggregates of amyloid-beta peptide, play a central role in the pathogenesis of Alzheimer's disease and represent a good target for treatment. We have shown previously that a 5-amino acid beta -sheet breaker peptide (iAbeta 5p), end-protected, has the ability to induce a dramatic reduction in amyloid deposition in two different transgenic Alzheimer's models (Permanne, B., Adessi, C., Saborio, G. P., Fraga, S., Frossard, M.-J., Dewachter, I., Van Dorpe, J., Banks, W. A., Van Leuven, F., and Soto, C. (2002) FASEB J. 16, 860-862). The aim of this study was to evaluate the effect of chemical modifications of the peptide bonds at the metabolite cleavage sites on the pharmacological properties of iAbeta 5p derivatives. Using a rational approach, peptide analogs were designed and tested for in vitro activity and enzymatic stability. One peptide analog containing a methyl group introduced at the nitrogen atom of one amide bond showed increased stability in vitro, a 10-fold higher in vivo half-life, and good brain uptake compared with iAbeta 5p while maintaining a similar activity in vitro. Our results suggest that the pharmacological profile of beta -sheet breaker peptides can be improved to produce compounds with drug-like properties that might offer a new promise in the treatment of Alzheimer's disease.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Alzheimer's disease is a degenerative disorder of the brain for which there is no cure or effective treatment. Recent studies suggest that cerebral amyloid plaques play a central role in the pathogenesis of the disease (1-3). An attractive therapeutic strategy for Alzheimer's disease is to block the early steps of misfolding and aggregation of the soluble amyloid-beta peptide (Abeta )1 (3). We proposed that short synthetic peptides capable of binding Abeta but unable to become part of a beta -sheet structure (beta -sheet breaker peptides) may destabilize the amyloidogenic Abeta conformer and hence preclude amyloid formation (3-5). A major drawback with the use of peptides as drugs in neurological diseases is their rapid metabolism by proteolytic enzymes and their poor blood-brain barrier (BBB) permeability (6-9). We reported previously that a 5-residue synthetic peptide (iAbeta 5, LPFFD) was able to inhibit and disassemble amyloid fibrils in vitro, to prevent Abeta neurotoxicity in cell culture, to arrest deposition of amyloid lesions, and to induce dissolution of preformed plaques in a rat brain model of amyloidosis (4, 5, 10). More recently, we showed that iAbeta 5 with N- and C-terminal protections to minimize exopeptidase cleavage (iAbeta 5p, Ac-LPFFD-NH2) was rapidly taken up by the brain and reduced in vivo amyloid deposition and cerebral damage in a double transgenic animal model of Alzheimer's disease (11). In addition, this compound showed low toxicity, low immunogenicity, and high solubility. The weakest aspect of this compound is its relatively short in vivo half-life.

The goal of this work was to introduce a series of chemical modifications into iAbeta 5p aiming to increase stability and simultaneously maintaining (or enhancing) potency, brain uptake, compound solubility, and low toxicity. A rational lead optimization strategy was followed by identification of the main metabolites of iAbeta 5p and design modifications at the catabolic sites of the sequence to prevent proteolytic degradation. Peptide analogs were screened by a set of in vitro and in vivo assays to select the most suitable compounds.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptide Synthesis-- iAbeta 5p and compounds in groups A-C were synthesized by Neosystem (Strasbourg, France); compounds 1, 2, and 5-8 in groups D and E were synthesized by Synt:em (Nîmes, France). All these compounds were synthesized in solid phase using Fmoc chemistry. Pseudoproline-containing compounds (D3 and D4) were synthesized at the Institute of Organic Chemistry (University of Lausanne, Lausanne, Switzerland) using Fmoc solid-phase chemistry as previously described (12). Peptides were purified by reverse-phase HPLC (RP-HPLC). Purity was >95% as estimated by RP-HPLC and mass spectrometric analysis.

Peptide Labeling-- Tritium-labeled compounds were synthesized by Amersham Biosciences (Buckinghamshire, UK). Labeling was incorporated during the synthesis using a 3,4-dehydroproline residue instead of proline at position 2. The dehydro-peptide was mixed with 10% palladium on calcium carbonate and dimethylformamide in a titration vessel. The mixture was stirred under tritium gas for 3 h. Thereafter, the solution was filtered, and labile tritium was removed by repeated evaporations to dryness with ethanol. The labeled peptide was purified by HPLC, and purity was estimated by mass spectrometry. Labeling at positions 3 and 4 of the Pro ring was considered chemically stable because <5% isotopic exchange with the solvent occurred in a period of 6 months. The specific activity determined by mass spectrometry was 75 Ci/mmol. Tritium radioactivity was measured by scintillation counting (Beckman Instruments).

In Vitro Assay of Peptide Stability-- Peptides were prepared as a 1 mM solution in phosphate-buffered saline. 20 µl of the peptide solution was diluted in 80 µl of human plasma (freshly taken) or 10% rat brain homogenate (in 1× phosphate-buffered saline and 0.5% Triton X-100). The solution was incubated at 37 °C for different times, and the reaction was stopped by adding the Complete mixture of protease inhibitors (Roche Molecular Biochemicals, Mannheim, Germany). The bulk of the plasma and brain proteins (but none of the peptide) were precipitated in cold methanol (1:4 (v/v) mixture/MeOH) for 1 h at -20 °C. The precipitated proteins were pelleted by centrifugation at 10,000 × g for 10 min at 4 °C. The supernatant containing the peptide was concentrated five times under vacuum and separated by RP-HPLC. The area of the peak (UV absorbance at 205 nm) corresponding to the intact peptide was measured and compared with an equivalent sample incubated in phosphate-buffered saline.

In Vitro Assay of Activity-- Amyloid formation was quantitatively evaluated by the fluorescence emission of thioflavin T bound to amyloid fibrils as reported previously (13, 14). Aliquots of Abeta -(1-42) at a concentration of 0.5 mg/ml prepared in 0.1 M Tris (pH 7.4) were incubated under gentle swirling for 5 days at 37 °C in the absence or presence of a 10-fold molar excess of the compounds. At the end of the incubation period, 50 mM glycine (pH 9.2) and 2 µM thioflavin T were added in a final volume of 2 ml. Fluorescence was measured at an excitation of 435 nm and an emission of 485 nm in a PerkinElmer Life Sciences Model LS50B fluorescence spectrometer.

Cellular Assay of Activity-- Aliquots of Abeta -(1-42) at a concentration of 0.5 mg/ml prepared in 0.1 M Tris (pH 7.4) were incubated alone or in the presence of different concentrations of the compounds for 36 h at 37 °C under gentle swirling. At the end of the incubation period, an aliquot of the solution was added to the medium of PC12 cells (Dulbecco's modified Eagle's medium from BIOSOURCE) to reach a final concentration of 5.5 µM Abeta . The cells were incubated for 24 h; and thereafter, the cellular viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide kit (Roche Molecular Biochemicals) according to the manufacturer's instructions.

Pharmacokinetic and Blood-Brain Barrier Permeability Studies-- Mice (CD-1, 28-36 g, in a house colony; Veterans Affairs Medical Center, Saint Louis, MO) were anesthetized with intraperitoneal urethane (40%), and the left jugular vein and the right carotid artery were exposed. 0.2 ml of Ringer's lactate solution (7.19 g/liter NaCl, 0.3 g/liter KCl, 0.28 CaCl2, 2.1 g/liter NaHCO3, 0.16 g/liter KH2PO4, 0.37 g/liter MgCl2·6H2O, 0.99 g/liter D-glucose, and 10 g/liter bovine serum albumin (pH 7.4)) containing 1% bovine serum albumin and the tritium-labeled peptide (100,000 cpm/ml) alone or with the unlabeled peptide was injected. Arterial blood was collected from the right carotid artery at different time points following intravenous injection into tubes containing the complete mixture of protease inhibitors. Serum was obtained by centrifugation at 4800 × g for 10 min at 4 °C. Following arterial blood collection, the mice were decapitated, and the whole brains (except the pineals and pituitaries) were harvested and weighed. The amount of radioactivity in the brain and serum was determined after an overnight solubilization step in TS-2 solution (Research Product International Corp., Mount Prospect, IL) at 40 °C.

The brain/serum ratio (µl/g) was estimated by the following equation: brain/serum ratio = (cpm/g of brain)/(cpm/µl of serum). The influx rate (Ki; µl of serum/g of brain/min) represents the rate at which compounds are moving from the circulation into the brain. The volume of distribution (Vi; µl of serum/g of brain) is the apparent volume of material that is distributed to the brain at time 0 and was estimated by extrapolation.

The amount of intact peptide contained in blood or brain samples was determined by HPLC analysis. Serum or brain homogenate (10%) containing the complete mixture of protease inhibitors was treated as described above under "In Vitro Assay of Peptide Stability."

To wash out blood from the brains, animals were perfused with 20 ml of Ringer's lactate solution (0.9% NaCl) in the left ventricle of the heart for 30 s. Capillary depletion studies were done as described (15). Briefly, the cerebral cortex was weighted and homogenized in physiological buffer (10 mM HEPES, 140 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1 mM MgSO4, 1 mM NaH2PO4, and 10 mM D-glucose (pH 7.4)). Dextran solution (1.6 ml of a 26% solution) was then added to the homogenate. After centrifugation at 5400 × g for 15 min at 4 °C, the pellet containing the brain vasculature and the supernatant containing the brain parenchyma were carefully separated, and the level of tritium was counted in each fraction.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Identification of Major Sites of Proteolytic Degradation in iAbeta 5p

In vivo pharmacokinetic parameters of iAbeta 5p were studied in mice by a single intravenous bolus injection of the tritium-labeled peptide into the tail veins. The half-life of the intact peptide determined by HPLC analysis of blood samples was 7 ± 3 min, consistent with the rapid clearance of the compound from the blood, as previously reported in rats (11). The HPLC analysis of the serum samples showed that the peptide was degraded into three main metabolites (Fig. 1A) with HPLC retention times of 30, 40, and 47 min, respectively. Incubation of iAbeta 5p with serum in vitro did not show any degradation after 24 h, as previously reported (11). However, the same peaks detected in blood after intravenous injection were observed upon incubation of iAbeta 5p in brain homogenate in vitro for 30 min at 37 °C, indicating that proteolytic degradation in vivo occurred in tissue, but not in blood. Three radioactive peaks named Ma, Mb, and Mc (retention times of 30, 40, and 47 min, respectively) were obtained, plus a fraction (fraction P) with retention identical to that of the intact peptide (retention time of 50 min). Analysis by mass spectrometry of the last fraction confirmed its identity to the intact peptide. Detection of proline-containing metabolites in the fractions of interest was done by precursor ion scanning tandem mass spectrometry. Scans performed using the mass of the immonium fragment ion of proline (m/z 70) allowed the detection of parent ions at m/z 271 and 418 in fractions Mb and Mc. The tandem mass spectra identified the sequences of the metabolites Ac-LP and Ac-LPF in these fractions (Fig. 1, B and C). Finally, the retention times of the chemically synthesized metabolites were identical to those of fractions Mb and Mc. Fraction Ma represents a heterogeneous peak that has not been identified. The two major metabolites found corresponded to proteolytic cleavage of the amide bonds between amino acids 2 and 3 (major cleavage) and between residues 3 and 4 (minor cleavage) (Fig. 1D).


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Fig. 1.   Identification of iAbeta 5p metabolites. Shown are the results from RP-HPLC analysis of serum samples obtained 20 min after injection of tritium-labeled iAbeta 5p in mice. The cpm in the eluate showed four radioactive fractions: Ma, Mb, Mc, and P (A). nd, not determined. Tandem mass spectrometric analysis of those fractions was performed to identify iAbeta 5p (fraction P) and its metabolites Mb and Mc (B and C, respectively). In fractions Mb (B) and Mc (C), the results from tandem mass spectrometric analysis indicate that the peaks at m/z 271.2 (fragments m/z 156.1, 116.1, 86.1, and 70.1) and at m/z 418.29 (fragments m/z 263.1, 253.16, 88.07, and 70.1) correspond to Ac-LP and Ac-LPF, respectively. A schematic representation of iAbeta 5p showing the peptide bonds where major cleavage sites were identified is presented (D).

Design of Peptide Analogs of iAbeta 5p

The strategy for lead optimization was to protect the peptide bonds involved in the proteolytic cleavage of the peptide by introducing chemical modifications around them (Fig. 1D). The following five groups of chemical modifications were studied for increased enzymatic stability of the peptide (Table I).


                              
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Table I
Schematic representation of the five groups of iAbeta 5p analogs studied
Boldface type indicates a chemical modification introduced into the sequence of iAbeta 5p. H, N terminus-free.

Alkylation of Amide Nitrogen-- Replacement of the hydrogen attached to the amide nitrogen with an alkyl group was reported to prevent endopeptidase degradation of the peptide bond into which it is incorporated (8, 16). Several combinations of single, double, and triple N-methylations were introduced in the sequence of iAbeta 5p.

Alkylation of alpha -Carbon-- Modifications of the alpha -carbon by replacing the hydrogen with an alkyl group is an approach also reported to decrease protease degradation of the peptide (8, 17, 18). A modified peptide was synthesized by adding methyl groups at the alpha -carbons corresponding to amino acids 3 and 4.

End Protection-- Most natural peptides with free N and C termini are degraded rapidly in blood and tissues by nonspecific exopeptidases (6, 8). Modification with chemical groups at both the N and C termini results in a lack of recognition by exopeptidases and a dramatic increase in peptide stability. A series of modifications were included at the N terminus of iAbeta 5p to determine the optimal chemical group for achieving the highest stability, solubility, and activity.

Non-natural Side Chains-- One approach for stabilizing a peptide is to modify the side chains of some of the amino acids involved in the protease recognition site. The residues of interest are replaced with non-natural amino acids with chemically similar side chains. The idea is to modify the recognition site of the enzyme to prevent the cleavage while staying as close as possible to the original sequence to preserve the activity of the peptide. In our case, the protease recognition site involves residues 2 and 3. Therefore, a series of modifications of the side chains of Pro and Phe were synthesized.

Cyclization-- The design of cyclic analogs represents another widely used strategy to increase peptide stability and potency (19). Cyclization confers a structural constraint that reduces conformational flexibility and may enhance potency, selectivity, stability, and bioavailability as well as membrane barrier permeability. To evaluate this approach, a cyclic peptide was synthesized by introducing 1 cysteine at each end and linking them by a disulfide bridge.

In Vitro Activity and Stability of iAbeta 5p Derivatives

First, all peptide derivatives were screened for in vitro activity in inhibition of amyloid fibril formation. Fibril formation was quantified with a fluorescence assay based on the specific binding of thioflavin T to beta -sheet amyloid aggregates (13, 14). The results are expressed in Fig. 2 as the percent inhibition of fibrillogenesis compared with the activity of the iAbeta 5p compound (100% activity). The following compounds had >80% activity: iAbeta 5p-A1, iAbeta 5p-A4, iAbeta 5p-A5, iAbeta 5p-A7, iAbeta 5p-B1, iAbeta 5p-C1, iAbeta 5p-C2, and iAbeta 5p-C3. This set of compounds was selected for evaluation of resistance to enzymatic degradation in vitro. All compounds in group D (modifications of proline or phenylalanine) and in group E (cyclization) showed dramatically reduced activities (<40% of the activity of iAbeta 5p).


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Fig. 2.   In vitro activity of the compounds in the fibrillogenesis assay. All compounds synthesized were screened for activity using the thioflavin T fluorescence assay for quantification of amyloid fibril formation by synthetic Abeta . Abeta -(1-42) (110 µM) in 0.1 M Tris (pH 7.4) was incubated alone or in the presence of a 10-fold molar excess of each of the compounds for 5 days at 37 °C. Thereafter, the amount of amyloid fibrils was measured using the thioflavin T fluorescence method as described under "Experimental Procedures." Values correspond to the percent inhibition of amyloid formation and are expressed relative to the activity of iAbeta 5p (100%).

The selected peptide analogs were incubated in vitro with human plasma or rat brain homogenate to model degradation in blood and tissues, respectively. The results shown in Table II indicate that the most stable compounds are iAbeta 5p-A1, iAbeta 5p-A5, iAbeta 5p-A7, and iAbeta 5p-B1. These compounds showed very little or no degradation during the 24-h study period (Table II).


                              
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Table II
In vitro stability of selected compounds
The biological stability of the unlabeled compounds was evaluated by incubating them (20 nmol) in fresh human plasma or 10% rat brain homogenate for different times at 37 °C. The amount of intact peptide was estimated by HPLC after removing the bulk of proteins by methanol precipitation. The values correspond to the in vitro half-life, which is defined as the time needed for 50% degradation of the peptide.

To further reduce the number of compounds to be tested in vivo, we used a cellular model of amyloid-induced cytotoxicity as a second screen for in vitro activity. Toxicity of Abeta in cell culture has been reported to be related to the formation of beta -sheet-rich aggregates (20, 21) and has been used to screen diverse compounds to prevent amyloid neurotoxicity. The results from this study indicate that iAbeta 5p-A1 has similar (or even greater) activity to iAbeta 5p in preventing amyloid-induced cell death (Fig. 3). A 10-residue unrelated peptide that was used as a control was found not to alter Abeta cytotoxicity. As part of our screening studies, we have used many different control peptides, including unrelated peptides of the same length, reverse and scrambled sequences, fragments containing the sequence of Abeta used as the template for iAbeta 5p, and beta -sheet breaker peptides designed to inhibit formation of beta -sheet aggregates in other systems (4, 5, 10). The results show that none of these peptides exhibit significant inhibitory activity and that even some of them enhance amyloid formation, most likely by incorporation into the aggregates (4, 5, 10). iAbeta 5p-A1 was selected as a prototype of a beta -sheet breaker peptide with similar in vitro activity to iAbeta 5p, but with much higher biological stability.


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Fig. 3.   In vitro activity of the compounds in a cellular assay of amyloid cytotoxicity. Abeta -(1-42) (110 µM) in 0.1 M Tris (pH 7.4) was incubated with different concentrations of iAbeta 5p (black-square), iAbeta 5p-A1 (black-diamond ), iAbeta 5p-A7 (triangle ), iAbeta 5p-B1 (open circle ), or an unrelated peptide (RSYPLPQG) used as a control (×). The solution was then diluted in cell culture medium and added to semiconfluent PC12 cells. Cells were incubated for 24 h, and cellular viability was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Values correspond to the percent cellular viability and represent the mean ± S.D. of four measurements. Treatment with Abeta -(1-42) alone under these conditions produced ~60% cell death (40% of the cells remained alive). Concentrations of the compounds correspond to the final concentrations in the cell culture medium.

In Vivo Pharmacokinetic Properties and Brain Uptake of the Lead Optimized Compound

The pharmacokinetic properties and BBB permeability of iAbeta 5p-A1 were studied in vivo and compared with those of iAbeta 5p. Fig. 4 shows the results of a pharmacokinetic study done in mice by a single intravenous bolus injection of either iAbeta 5p or iAbeta 5p-A1. The latter compound has a 10-fold greater half-life (70 min versus 7 min) and a >70-fold higher area under the curve compared with iAbeta 5p.


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Fig. 4.   Pharmacokinetic study of iAbeta 5p and iAbeta 5p-A1. The serum concentration of intact tritium-radiolabeled iAbeta 5p (black-square) or iAbeta 5p-A1 (black-diamond ) after intravenous administration to mice was estimated by HPLC as described under "Experimental Procedures." The half-life was calculated from the linear part of the curve.

The ability of iAbeta 5p-A1 to cross the BBB was analyzed by brain perfusion of the tritium-labeled peptide. Radioactivity corresponding to the intact peptide was rapidly observed in the brain as analyzed by HPLC. To determine whether the peptide has effectively penetrated into the brain parenchyma, a capillary depletion experiment was performed. 10 min after injection of tritium-labeled peptide, >70% of the radioactivity was recovered in the parenchyma (data not shown). The percent radioactivities associated with the capillary fraction with and without blood washout were 24 and 29%, respectively. This result indicates that the majority of the compound in the brain had effectively crossed the BBB and that the peptide did not bind to the luminal surface of the capillaries and was not greatly sequestrated by the brain endothelial cells.

To estimate the amount of compound entering the brain, mice were injected intravenously with the radioactive peptide. The percentage of the injected dose of compound taken up per g of brain was calculated. The maximum radioactivity value was 0.464%, and half of this value was reached 1.4 min after injection (Fig. 5). These data suggest rapid brain uptake of iAbeta 5p-A1. HPLC analysis of brain samples taken 20 min after the intravenous injection showed that 12% of the radioactivity corresponded to the intact peptide. Therefore, we calculated that >0.06% of the injected compound was recovered intact in the brain within the first 20 min after injection. These data are similar to what was found previously for iAbeta 5p, for which an average of 0.09% of the injected peptide reached the brain in an intact form (11).


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Fig. 5.   BBB permeability study of iAbeta 5p-A1 in mice. The percentage of the intravenously injected dose of peptide taken up per g of brain was calculated as described under "Experimental Procedures."

To understand the mechanism of brain uptake, a mixture of labeled and unlabeled peptide was injected. The brain/serum ratio of total radioactivity was determined between 1 and 120 min of exposure. The influx rate (Ki) with the radioactive peptide alone was 3.54 ± 0.29 µl/g/min, and the volume of distribution (Vi), calculated from the y intercept, was ~50 µl/g (Table III). Brain uptake was inhibited by injection of unlabeled material. 32 and 52% statistically significant decreases in Ki (p = 0.006) were observed after injection of 10 and 100 µg of nonradioactive peptide/mouse, respectively. This result indicates that iAbeta 5p-A1 penetrates the brain through a saturable mechanism.


                              
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Table III
Brain uptake of iAbeta 5p-A1
Tritium-labeled iAbeta 5p-A1 alone (labeled) or mixed with unlabeled peptide (10 and 100 µg/mouse) was injected intravenously into mice. The animals were killed, and serum and brain samples were taken after different exposure times (1-120 min). Kinetic brain uptake parameters (Ki, influx rate; Vi, volume of distribution) were measured.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peptides are amenable to the process of rationale drug development. It is relatively easy to develop peptides with in vitro activity, high selectivity, and low toxicity (22). However, due to their predisposition for enzymatic degradation, unmodified peptides do not circulate in blood for more than a few minutes (6). Moreover, they have generally poor bioavailability in tissues and organs, preventing their usefulness as therapeutic agents. Therefore, to use peptides as a drug, they have to be chemically modified so that proteolytic degradation is diminished and bioavailability is increased. The impressive advance in the field of peptide solid- and liquid-phase synthesis allows the introduction of a wide variety of chemical modifications into a peptide backbone (8, 23). These modifications are useful only if they are strategically introduced to maximize enzymatic stability and bioavailability and simultaneously preserve or enhance potency and selectivity of the bioactive peptide. This approach requires knowledge of the major in vivo proteolytic sites of the peptide and ideally the chemical groups in the molecule responsible for its biological activity (6, 9, 24).

The aim of this study was to analyze and improve the pharmacological properties of a 5-residue beta -sheet breaker peptide active in reducing amyloid load and cerebral damage in animal models of Alzheimer's disease. beta -Sheet breaker peptides are short synthetic peptides homologous to the central fragment of Abeta containing residues that destabilize beta -sheet structures (3). Sequence similarity is important for the specific interaction of beta -sheet breaker peptides with Abeta . The introduction of Pro as a beta -sheet blocker residue is important for destabilizing intermolecular beta -sheets of Abeta aggregates (12, 25). Our approach for lead optimization was to stay as close as possible to the original sequence of iAbeta 5p to preserve activity and selectivity, introducing only strategic chemical modifications to protect the peptide against proteolytic degradation.

Two major cleavage sites were found to be located at the peptide bonds between Pro and Phe and between the two phenylalanines. Interestingly, these amino acid sequences are known to be recognition sites for proteolytic degradation of some endogenous neuropeptides such as bradykinin and substance P (6, 26) by peptidases such as dipeptidyl peptidase IV, endopeptidase-24.11, and angiotensin-converting enzyme (27, 28). Indeed, we found that phenelzine, an inhibitor of angiotensin-converting enzyme, reduced the kinetics of iAbeta 5p degradation in vitro in rat brain homogenate (data not shown). In addition, several endo- and exopeptidases are specific for cleavage of peptides after Pro (29, 30), and it has been proposed that this is critical in controlling the turnover of important proteins containing Pro-rich domains (29). We hypothesized that these endopeptidases found in plasma membranes of a variety of cells, including brain (31), may be responsible for the degradation of iAbeta 5p in vivo.

Chemical modifications such as methylations of the amide bond nitrogen and of the alpha -carbon introduced at the cleavage sites of iAbeta 5p improved significantly the enzymatic stability of these analogs in rat brain homogenate. The major protective effect was observed when these modifications were introduced at the peptide bond between Pro and Phe, supporting the finding that this is the major cleavage site of iAbeta 5p. Some of these compounds, including iAbeta 5p-A1 (single N-methylation between Pro and Phe), were similarly or even more active than the unmodified peptide in both the fibrillogenesis and cellular toxicity assays. Interestingly, it has been shown that short Abeta fragments around sequence 16-21 and N-methylated at alternate positions are able to inhibit amyloid fibril formation in vitro (32). Surprisingly, the compound containing a double methylation at the alpha -carbons (iAbeta 5p-B1) was active in the fibrillogenesis assay, but less active in the cellular toxicity assay. The reason for this result is under investigation.

Although chemical modification at the amide backbone resulted in some active compounds, the replacement of Pro and Phe with non-natural derivatives dramatically decreased activity in vitro in all derivatives. These results suggest that Pro and Phe at positions 2 and 3, respectively, play a fundamental role in the activity of beta -sheet breaker peptides. It has been shown previously by several groups that Abeta sequence 17-21 and in particular Phe19 (equivalent to Phe3 of iAbeta 5p) are critical for Abeta -Abeta interaction and amyloid formation (13, 25, 33, 34). It is likely that the phenylalanines (positions 3 and 4) and leucine (position 1) are responsible for selective binding of the beta -sheet breaker peptides to Abeta . On the other hand, the Pro residue does not seem to play a role in binding, but it is critical for activity. Indeed, replacement of Pro with non-beta -sheet blocker amino acids such as alanine and valine did not decrease binding of iAbeta 5p to Abeta , but dramatically reduced the ability of the compound to inhibit amyloid formation in vitro (data not shown). Pro acts as a beta -sheet blocker due to its constraint to fold in a beta -sheet structure (12, 25, 35). These results suggest the importance of maintaining a key balance between sequence homology of Abeta and the beta -sheet breaker peptide and the presence of amino acids able to disrupt beta -sheet folding. Complete sequence homology produces a compound with the highest affinity for Abeta , but which is unable to structurally destabilize beta -sheets and thus becomes incorporated into amyloid aggregates. Compounds with too many beta -sheet blockers have a reduced affinity for Abeta and hence lower potency.

A unique characteristic of Pro is its ability to interconvert between cis- and trans-configurations (36, 37). To evaluate the influence of cis/trans-switching on the activity of beta -sheet breaker peptides, we synthesized derivatives containing pseudoprolines in place of Pro. Pseudoprolines were shown to enhance the Pro features by stabilizing the cis-form of the peptide (36). In addition, depending on the chemical groups attached to the pseudoproline ring, it is possible to generate compounds with different cis/trans ratios (36). The results obtained with the two pseudoproline-containing peptides (iAbeta 5p-D3 and iAbeta 5p-D4) indicate that stabilization of the cis-configuration decreased the activity of the peptide, suggesting that Pro in the trans-conformation is required for activity. An alternative possibility is that cis-Pro is the active conformation, but that trans-Pro is required for binding to the target. The latter possibility suggests that conformational flexibility of beta -sheet breaker peptides might be important for activity. More studies are necessary to investigate these possibilities.

A critical requirement for the use of beta -sheet breaker peptides in the treatment of amyloidosis in Alzheimer's disease is their ability to enter the brain. We had already reported evidence for high penetration of iAbeta 5p across the BBB (11). Brain uptake of iAbeta 5p-A1 is similar to that previously shown for iAbeta 5p. The percentage of intact iAbeta 5p-A1 crossing the BBB is in the range of substances known to exert an effect in the brain such as interleukin-1 and morphine (0.08 and 0.02%, respectively) (38) and certainly in the upper range of permeability expected for peptide molecules (from 0.01 to 0.1%). Our results indicate that iAbeta 5p-A1 crosses the BBB by a saturable mechanism, most likely a receptor-mediated transport. At present, the endogenous ligand of the transporter and the nature of the receptor are unknown. In conclusion, our results suggest that the pharmacological profile of peptides can be improved and that application of some of these strategies to beta -sheet breaker peptides might result in compounds useful in the treatment of Alzheimer's disease.

    ACKNOWLEDGEMENTS

We thank MScan SA (Geneva, Switzerland) for mass spectrometry analysis, Serge Halazy, Youcef Fezoui, and John Lopez for valuable contributions on peptide design and synthesis, and Kinsey Maundrell for critical reading of the manuscript.

    FOOTNOTES

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|| To whom correspondence should be addressed: Serono Pharmaceutical Research Inst., Chemin des Aulx, 14, Plan les Ouates, 1228 Geneva, Switzerland. Tel.: 41-22-739-3944; Fax: 41-22-739-3033; E-mail: Claudio.Soto@serono.com.

Published, JBC Papers in Press, February 10, 2003, DOI 10.1074/jbc.M211976200

    ABBREVIATIONS

The abbreviations used are: Abeta , amyloid-beta peptide; BBB, blood-brain barrier; Fmoc, N-(9-fluorenyl)methoxycarbonyl; RP-HPLC, reverse-phase high pressure liquid chromatography.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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