From the 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
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ABSTRACT |
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Amyloid plaques in brain, composed of aggregates
of amyloid- 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- The goal of this work was to introduce a series of chemical
modifications into iA Peptide Synthesis--
iA 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 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
A Cellular Assay of Activity--
Aliquots of A 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.
Identification of Major Sites of Proteolytic Degradation in
iA In vivo pharmacokinetic parameters of iA 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
-sheet breaker
peptide (iA
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 iA
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
iA
5p while maintaining a similar activity in vitro. Our
results suggest that the pharmacological profile of
-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
peptide
(A
)1 (3). We proposed that
short synthetic peptides capable of binding A
but unable to become
part of a
-sheet structure (
-sheet breaker peptides) may
destabilize the amyloidogenic A
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 (iA
5, LPFFD)
was able to inhibit and disassemble amyloid fibrils in
vitro, to prevent A
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 iA
5 with N- and C-terminal protections to minimize
exopeptidase cleavage (iA
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.
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
iA
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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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.
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.
-(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.
-(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 A
. 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.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
5p
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
iA
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 iA
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).
View larger version (18K):
[in a new window]
Fig. 1.
Identification of
iA 5p metabolites. Shown are the results
from RP-HPLC analysis of serum samples obtained 20 min after injection
of tritium-labeled iA
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 iA
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 iA
5p showing the peptide bonds where major
cleavage sites were identified is presented (D).
Design of Peptide Analogs of iA5p
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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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
iA5p.
Alkylation of -Carbon--
Modifications of the
-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
-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 iA5p 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 iA5p 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 -sheet amyloid aggregates (13,
14). The results are expressed in Fig. 2
as the percent inhibition of fibrillogenesis compared with the activity
of the iA
5p compound (100% activity). The following compounds had
>80% activity: iA
5p-A1, iA
5p-A4, iA
5p-A5, iA
5p-A7,
iA
5p-B1, iA
5p-C1, iA
5p-C2, and iA
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 iA
5p).
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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 iA5p-A1, iA
5p-A5, iA
5p-A7, and iA
5p-B1. These
compounds showed very little or no degradation during the 24-h study
period (Table II).
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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 A in
cell culture has been reported to be related to the formation of
-sheet-rich aggregates (20, 21) and has been used to screen diverse
compounds to prevent amyloid neurotoxicity. The results from this study indicate that iA
5p-A1 has similar (or even greater) activity to
iA
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 A
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 A
used as the
template for iA
5p, and
-sheet breaker peptides designed to
inhibit formation of
-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).
iA
5p-A1 was selected as a prototype of a
-sheet breaker peptide
with similar in vitro activity to iA
5p, but with much higher biological stability.
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In Vivo Pharmacokinetic Properties and Brain Uptake of the Lead Optimized Compound
The pharmacokinetic properties and BBB permeability of iA5p-A1
were studied in vivo and compared with those of iA
5p.
Fig. 4 shows the results of a
pharmacokinetic study done in mice by a single intravenous bolus
injection of either iA
5p or iA
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 iA
5p.
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The ability of iA5p-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 iA5p-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 iA
5p, for
which an average of 0.09% of the injected peptide reached the brain in
an intact form (11).
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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 iA5p-A1 penetrates the brain through a saturable
mechanism.
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DISCUSSION |
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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 -sheet breaker peptide active in reducing
amyloid load and cerebral damage in animal models of Alzheimer's
disease.
-Sheet breaker peptides are short synthetic peptides
homologous to the central fragment of A
containing residues that
destabilize
-sheet structures (3). Sequence similarity is important
for the specific interaction of
-sheet breaker peptides with A
.
The introduction of Pro as a
-sheet blocker residue is
important for destabilizing intermolecular
-sheets of A
aggregates (12, 25). Our approach for lead optimization was to stay as close as possible to the original sequence of iA
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 iA5p 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 iA
5p
in vivo.
Chemical modifications such as methylations of the amide bond nitrogen
and of the -carbon introduced at the cleavage sites of iA
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 iA
5p.
Some of these compounds, including iA
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 A
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
-carbons
(iA
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 -sheet
breaker peptides. It has been shown previously by several groups that
A
sequence 17-21 and in particular Phe19 (equivalent to
Phe3 of iA
5p) are critical for A
-A
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
-sheet breaker peptides to
A
. 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-
-sheet blocker amino acids such
as alanine and valine did not decrease binding of iA
5p to A
, but
dramatically reduced the ability of the compound to inhibit amyloid
formation in vitro (data not shown). Pro acts as a
-sheet
blocker due to its constraint to fold in a
-sheet structure (12, 25,
35). These results suggest the importance of maintaining a key balance
between sequence homology of A
and the
-sheet breaker
peptide and the presence of amino acids able to disrupt
-sheet
folding. Complete sequence homology produces a compound with the
highest affinity for A
, but which is unable to structurally
destabilize
-sheets and thus becomes incorporated into amyloid
aggregates. Compounds with too many
-sheet blockers have a reduced
affinity for A
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 -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 (iA
5p-D3 and
iA
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
-sheet breaker peptides might be
important for activity. More studies are necessary to investigate these possibilities.
A critical requirement for the use of -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 iA
5p across the BBB (11). Brain uptake of iA
5p-A1 is similar
to that previously shown for iA
5p. The percentage of intact
iA
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 iA
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
-sheet breaker peptides
might result in compounds useful in the treatment of Alzheimer's disease.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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
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ABBREVIATIONS |
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The abbreviations used are:
A, amyloid-
peptide;
BBB, blood-brain barrier;
Fmoc, N-(9-fluorenyl)methoxycarbonyl;
RP-HPLC, reverse-phase high pressure liquid chromatography.
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REFERENCES |
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