Reversal of aggregation using β-breaker dipeptide containing peptides: Application to aβ(1-40) self-assembly and its inhibition

Article abstract of DOI:10.1021/cn500064z

Reversion of protein or peptide aggregation is a formidable task, important in various domains of research at the interface of chemistry, medicine, and nanoscience. A novel class of dipeptides, termed as β-breaker dipeptides (BBDPs), is identified, which can be incorporated into the self-recognizing sequences to generate a novel class of conformational switch which forms β-sheet at an initial stage and then converts in a controlled manner to random coil at specific conditions. Incorporation of BBDPs in a well designed amyloidogenic peptides generates a special class of β-sheet breaker peptides those undergo a chemical change at physiological condition generating a breaker element in situ. These β-breaker peptides are shown to first incorporate into the amyloid and then disrupt it. Such conformational switches may be used to study agrregation/disaggregation process and may find many biomedical applications relevant to aggregation related disorders. Such strategy for reversion of peptide aggregation using chemical tricks may find application in material chemistry as well.

Full text of DOI:10.1021/cn500064z

Research Article
pubs.acs.org/chemneuro
Reversal of Aggregation Using β‑Breaker Dipeptide Containing
Peptides: Application to Aβ(1−40) Self-Assembly and Its Inhibition
Krishna Chaitanya Nadimpally, Ashim Paul, and Bhubaneswar Mandal*
Laboratory of Peptide and Amyloid Research, Department of Chemistry, Indian Institute of Technology Guwahati, Assam-781039,
India
S
* Supporting Information
ABSTRACT: Reversion of protein or peptide aggregation is a formidable
task, important in various domains of research at the interface of
chemistry, medicine, and nanoscience. A novel class of dipeptides, termed
as β-breaker dipeptides (BBDPs), is identified, which can be incorporated
into the self-recognizing sequences to generate a novel class of
conformational switch which forms β-sheet at an initial stage and then
converts in a controlled manner to random coil at specific conditions.
Incorporation of BBDPs in a well designed amyloidogenic peptides
generates a special class of β-sheet breaker peptides those undergo a
chemical change at physiological condition generating a breaker element
in situ. These β-breaker peptides are shown to first incorporate into the
amyloid and then disrupt it. Such conformational switches may be used to
study agrregation/disaggregation process and may find many biomedical
applications relevant to aggregation related disorders. Such strategy for reversion of peptide aggregation using chemical tricks
may find application in material chemistry as well.
KEYWORDS: Alzheimer’s disease, self-assembly, β-sheet, Aβ(1−40) peptide
peptides can disrupt existing amyloid also.4b But adequate
ggregation and disaggregation of proteins and peptides are
literature on the mechanism of disruption is unavailable.8 Many
developments are observed for improving solubility and
stability of such peptides,9 but the basic structure of such
peptides was always kept unaltered.
Aat the center of interest in various subjects spanning from
nanosciences, membrane technology, gelation, drug delivery to
drug discovery, and medicine. Amyloidoses, such as Alzheimer’s
disease (AD), Parkinson’s disease, Huntington’s disease,
diabetes type II, prion disorders, and so forth, are characterized
by the aggregation of peptides or proteins of variable length and
amino acid composition.1 Growing evidence suggests that the
self-assembly of amyloid β (Aβ), a 39−42-residue peptide, into
amyloid fibrils is the main cause of Alzheimer’s disease.2 Aβ
peptide, which usually exists in soluble random coil or helix
conformation in its native state, undergoes misfolding to form
insoluble composites rich in ordered aggregates of stacked β-
sheet structures, which is most stable. Low molecular weight
aggregates, which are soluble and thus able to migrate, are
believed to be more toxic than insoluble aggregates.3 Use of β-
breaker peptides, first introduced by Soto et al., is a landmark in
the direction of fibrillization inhibitory peptide based drug
design.4 Soto et al. prepared β-breaker peptides containing
proline as the β-breaker element attached with a recognition
sequence. Another promising approach is the N-methylation of
certain residues of the Aβ recognizing sequence, which
interferes with the hydrogen bonding networks, thus preventing
β-sheet stacking.5 α-Aminoisobutyric acid (Aib) is also known
to be more efficient than proline as β-sheet destabilizing
element.6 Aib as a β-breaker element was used to inhibit the
aggregation of human IAPP, a 37 residue peptide responsible
for type II diabetes.7 It is reported that these β-sheet breaker
Close analysis of the structure of the existing β sheet breaker
peptides reveals that all of them mainly consist of a breaking
element (for example, a kink) and a recognition element (the
rest of the peptide), where the breaking element was
preinstalled. Recognition or binding affinity is governed by
the number of H-bonding possibilities, hydrophobic inter-
actions, and structural fit into the planar β-sheet topology. On
the contrary, breaking effect is achieved by reducing the
number of H-bonds, reducing the hydrophobic nature and
introducing a group which can not fit structurally in the planar
topology of β-sheet. Thus, “recognition” and “breaking” is in
contrast to each other. Insertion of chemical entity in a breaker
peptide to improve the disruption phenomenon results in
reduction of recognition efficiency and vice versa. Thus, the
preinstalled disrupting element, for example, kink (for proline
and Aib) or N-methylated amino acids, may lessen the chance
of proper alignment and recognition to the aggregating peptide
precluding the possibility of the β-breaking effect. Therefore,
design of β-sheet breaker peptides which can bind to either
Received: March 24, 2014
Published: March 25, 2014
© 2014 American Chemical Society
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Figure 1. Concept of β-sheet disruption using β-breaker-dipeptide containing β-sheet breaker peptides.
soluble or insoluble aggregates and exert breaking effect by in
situ generated breaking element is highly desirable.
feature of the BBDP concept is that a BBDP-containing β-sheet
breaker peptide readily and properly aligns with the Aβ peptide
(or any other aggregating peptide) if the sequence homology is
maintained. As the breaker element is not formed at the initial
stage, the problem of recognition between the breaker peptide
and the aggregating peptide is ruled out. When the pH of the
medium is adjusted to near physiological conditions, the BBDP
unit of the BBDP-containing β-sheet breaker peptide undergoes
a chemical transformation. That chemical transformation
generates the breaker element in situ, resulting in a kink into
the peptide backbone. At this stage, it can disrupt β-sheet or
fibril accumulation of itself as well as of the neighboring
aggregating peptide(s) when mixed (Figure 1).
Our concept is based on the chemistry of aspartimide
formation, a well-known side reaction frequently observed in
peptide chemistry.10 A BBDP forms aspartimide at neutral to
basic pH and around 37 °C temperature. Aspartimide induces a
type II′ β turn when incorporated into a peptide, which
generates a kink and disturbs the β-sheet topology of the
peptide.11 Moreover, ring-opening of the aspartimide residue
generates α- and β-aspartyl residues (in 1:3 ratio) at the
mentioned pH and temperature. The β-aspartyl derivative
introduces conformational flexibility in the system and breaks
down the hydrogen bonding network between the BBDP-
containing β-sheet breaker peptide and the aggregating peptide.
Furthermore, racemization of the Asp residue during the
process of aspartimide formation also contributes to the
disruption of the H-bonding network of the β-sheet.12 12.5%
of the total breaker peptide may convert back to the α aspartyl
peptide bearing -(L)Asp-Axx-, which may reinforce the effect of
To achieve the mentioned target, we have developed a novel
concept termed as “the concept of β-breaker dipeptides”
(BBDPs). A specially designed novel class of dipeptides is at
the center of the concept. When such a dipeptide is
incorporated at the middle of an aggregating peptide, it
generates a novel class of smart β-sheet breaker peptides, which
first forms β-sheet and changes back to random coil
conformation on prolonged treatment at basic pH. Kinetics
of the conformational change can be controlled by either
controlling the pH or altering the chemical structure. This
concept may have application across the domain. For example,
in the context of peptide based drug design against
amyloidoses, a new classs of β-sheet breaker peptides can be
designed for which the breaker element may be generated in
situ and problems of preinstalled breaker element can be
bypassed (Figure 1).
A dipeptide of the general formula [Asp(OP)-Axx] can act as
a β-breaker dipeptide unit. “P” stands for a suitable side chain
protecting group of an aspartic acid residue, and Axx can be any
amino acid. Conceptually, “Asp” can also be replaced by “Glu”
or any artificial amino acid bearing a carboxylic acid group on a
suitable position of the side chain. A novel class of BBDP-
containing β-sheet breaker peptides can be generated by
incorporating a suitable β-breaker dipeptide inside the self-
recognizing key sequence of the Aβ peptide (e.g., Aβ17−21),
which would be effective for disruption of Aβ aggregation.
Similar BBDP-containing β-sheet breaker peptides can also be
designed for other relevant aggregating peptides. The unique
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other in situ generated breaker peptides due to decrement of
the hydrophobic nature of the parent peptide via generation of
the negative charge on the side chain of Asp. Thus, complete
breakdown of the β-sheet architecture and amyloid may be
achieved using such a technique.
Conformational changes of all the breaker peptides were
monitored by Fourier transform infrared (FT-IR) spectroscopy
and circular dichroism (CD). In FT-IR spectroscopy, we
noticed the presence of a strong band at 1625 cm−1 for peptide
1 and 1626 cm−1 for peptide 2 incubated in sodium acetate
buffer for 4 days, indicating the amide I band of the peptide in
β-sheet conformation. On the other hand, after incubation for 4
days in PBS at pH 7.0, the same peptides revealed bands at
1645 and 1640 cm−1, respectively, which were assigned to the
amide I band of the peptide in random coil conformation
(Figure 2c).
RESULTS AND DISCUSSION
■
Peptides Used for the Studies. To test our hypothesis,
we first designed four β-breaker peptides (peptides 1−4 in
Table 1) in which the [Asp(OBzl)-Axx] unit was incorporated
The β-breaker peptides exhibited characteristic signals
corresponding to β-sheets when incubated in sodium acetate
buffer pH 4.0 for 4 days in their CD profiles. On the other
hand, when pH was regulated to neutral range, these peptides
underwent chemical modification and converted to the random
coil conformation (Supporting Information Figures S14 and
S15). When the solid sample of peptide 1 was dissolved in PBS
(pH 7) and measured, the CD profile exhibited a maximum at
195 nm and minimum at 220 nm, characteristic of β-sheet
conformation (Figure 2d) at the initial stage. When the same
sample was checked next day, the characteristic curve of a β-
turn was observed, that was converted to a characteristic curve
of a random coil after two more days. The identity of the
peptide, whether it is in α-aspartyl form, aspartimide form, or β-
aspartyl form, was confirmed by ESI-MS at specific time points.
The pH at which chemical conversion takes place depends on
the side chain protection of Asp and the following amino acid
(Axx) of the BBDP unit.11 For example, peptide 1 was observed
(both via ESI-MS and CD) to stay in α-aspartyl form (β-sheet)
up to 7 days in pH 4 (acetate buffer), whereas it was in α-
aspartyl form (β-sheet) at pH 7 (PBS) up to 12 h, which then
converted to aspartimide (β-turn) and aspartyl derivatives
(random coil). Thus, kinetics of the generation of the breaking
element in the newly proposed β-sheet breaker peptides can be
controlled by altering the BBDP unit. The amyloidogenic
nature of these peptides was studied using thioflavin T
fluorescence, transmission electron microscopy (TEM), and
Congo red birefringence. BBDP-containing peptides formed
amyloid-like fibrils in acidic buffer (Figure 2e, left panel), and
no such fibrillar aggregates were observed when incubated in
PBS (Figure 2e, right panel) and viewed under TEM after 4
days of incubation.
Also a characteristic green-gold birefringence was noticed
when peptide 1 was incubated in sodium acetate buffer for 4
days, stained with Congo red and observed under polarized
light (Figure 2f, left panel), indicating the presence of amyloid.
No birefringence was noticed when the same peptide was
incubated in PBS pH 7.0 for same time period of incubation
(Figure 3f, right panel). These results support our hypothesis
that O to N acyl migration at pH 7.0 leads to the formation of
aspartimide, and subsequent ring-opening of the aspartimide
generates α- and β-aspartyl derivatives of the BBDP-containing
β-sheet breaker peptide that completely inhibit the self-
assembly of the amyloidogenic peptides.
Increase in fluorescence intensity upon binding to thioflavin
T is considered as a characteristic property of fibrils and
therefore kinetics of fibril formation was assessed using this
technique.14 Strong enhancement of the thioflavin T
fluorescence emission intensity in presence of BBDP-
containing β-sheet breaker peptides at the initial stages of the
experiment (up to 24 h) followed by the gradual decrease of
the fluorescence over time revealed that the BBDP-containing
Table 1. Sequences of the Peptides Synthesized and Their
Role in the Current Study
peptide
no.
role of the
peptide
sequence
1
2
3
4
5
6
7
8
9
SLSD(OBzl)SLS-GNH2
LSD(OBzl)SLS-GNH2
Ac-SLSLHQKLD(OBzl)FFAEDVSL-GNH2
Ac-LD(OBzl)FFD-NH2
Ac-LPFFD-NH2
breaker
breaker
breaker
breaker
control
SLSL(H+)SLS-GNH2
aggregating
aggregating
control
Ac-SLSLHQKLVFF(H+)SEDVSL-GNH2
SLSPSLSGNH2
SLSDSLSGNH2
control
into the middle of the sequence. Additionally, peptide 5,
popularly known as Soto’s breaker peptide, was also
synthesized and used as a control. The BBDP unit was
composed of -D(OBzl)S- in peptides 1 and 2, and -D(OBzl)F-
in peptides 3 and 4. In all the peptides, “P” was chosen to be
“-Bzl”.
The sequences of these peptides were designed following the
concept of Hecht et al. of alternating hydrophilic and
hydrophobic amino acids13 (for peptides 1 and 2), and picking
the hydrophobic region of Aβ peptide (for peptides 3 and 4),
so that they form β-sheet and fibrils readily at the initial stage.
Once pH is altered from acidic to basic range, the BBDP unit
generates aspartimide and subsequently aspartyl derivatives of
the peptides, which results in disruption of self-aggregation.
Peptides 6 and 7 were used as model aggregating peptide or
functional mimic of Aβ peptide. Peptides 8 and 9 were used as
control peptide. In addition to that, commercially available
Aβ(1−40) peptide was also used. Sequence homology was
maintained among peptides 1, 2, 6, 8, and 9 for proper
comparison. Similarly, sequence homology was maintained
between peptides 3 and 7, as well as between peptides 4 and 5.
Reversion of Self-Aggregation of BBDP Containing
Model Peptides. The chemical conversion of the BBDP unit
of the breaker peptides to aspartimide and further ring-opening
to aspartyl residues (α:β = 1:3) was monitored by liquid
chromatography−mass spectrometry (LC−MS) in a time
dependent manner at pH 7.0 for peptides 1−3 and pH 7.4
for peptide 4 (see Figure 2a for peptide 1 and Figure 5a for
peptide 4; Supporting Information Figure S11 for peptide 2 and
Figure S12 for peptide 3). In general, conversion to aspartimide
took 24 h and conversion of that to α- and β-aspartyl peptides
took 48 h. Stability of the BBDP unit of peptide 1 in sodium
acetate buffer (pH 4.0) was also examined, and no acyl
migration was noticed even after 1 week (Figure 2b). Next, we
investigated the ability of the BBDP-containing β-sheet breaker
peptides to inhibit/disrupt self-aggregation.
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Figure 2. (a) LC−MS monitoring of aspartimide formation and subsequent hydrolysis of BBDP-containing peptide 1. (b) LC−MS profile of peptide
1 incubated in sodium acetate buffer after 1 week, indicating stability. (c) FT-IR spectra of peptide 1 and 2 incubated in pH 4 and PBS pH 7.0. (d)
CD spectra of peptide 1 in PBS (pH 7.0). (e) TEM images of peptide 1 incubated in sodium acetate buffer (left panel) and PBS pH 7.0 (right
panel), showing the presence and absence of fibrils, respectively, after 4 days of incubation. (f) Congo red birefringence image of peptide 1 incubated
in acidic (left panel) and basic buffer (right panel), indicating the presence and absence of amyloid, respectively, after 4 days of incubation. (g) Time
dependent studies of fibril evolution using thioflavin T fluorescence experiment at pH 7.0 (black curve for peptide 1 and red curve for peptide 2).
peptides first aggregate to form amyloid-like fibrils and then
slowly disrupt that (Figure 2g). Kinetics of O to N acyl
migration revealed by LC−MS studies (Figure 2a) also
demonstrates that peptide 1 takes about 24 h for complete
conversion to either aspartimide or the aspartyl derivatives.
Such concordance of the kinetics of rearrangement, aggrega-
tion, and disruption was noted for all other BBDP containing
peptides as well, which indicates that fibril disruption was
caused by in situ aspartimide formation and subsequent ring-
opening. Results mentioned above demonstrates that BBDP
containing peptides first forms β sheet and fibril then self-
aggregation is disrupted slowly by specific chemical conversion
at specific pH range. To the best of our knowledge, this is the
first report of a model aggregating peptide, which first
aggregates to form fibril and then disintegrates by itself via a
chemical reaction in a controlled manner. Such system can be
useful for studies of the process of aggregation and
disaggregation.
Since there is a possibility of coaggregation of such BBDP-
containing β-sheet breaker peptides with neighboring aggregat-
ing peptides, for example, Aβ peptide when mixed, we
hypothesized that a suitably designed BBDP-containing β-
breaker peptide can disrupt the Alzheimer’s Aβ amyloid when
coincubated in an appropriate proportion with Aβ peptide.
However, synthesis and handling of the full length Aβ peptide
is difficult. Therefore, we thought if we have a system that is
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Figure 3. (a) pH induced O to N acyl migration of peptide 6 (left) and 7 (right). (b) CD spectra of peptide 6 and 7 in sodium acetate buffer (pH
4.0) and PBS (pH 7.0) buffer after 4 days of incubation. (c) TEM micrographs of peptide 6 and 7 at various pH after four days of incubation.
Figure 4. Time dependent thioflavin T fluorescence measurement at pH 7.0 of (a) peptide 6 in the absence (black) and presence (red) of the
breaker peptide 1; (b) peptide 7 in the absence (black) and presence (red) of breaker peptide 3; (c) peptide 6 in the absence (black) and presence of
the control peptide 8 (red), 9 (green), the breaker peptide 1 (blue).
easily accessible and behaves similar to the full length Aβ
peptide (Aβ mimic), it would be easy to test the hypothesis
before testing it on the full length Aβ.
in a regular peptide longer enough to form β-sheet as well as
fibrils. Along a similar line, a host−guest switch peptide was
also reported where the hydrophobic core sequence of the Aβ
peptide was embedded inside the -(Ser-Leu)n- based
aggregating sequence containing a serine-based switch at a
suitable position.15b
We have chosen a -(Ser-Leu)n- based switch peptide bearing
the sequence SLSL(H+)SLS-GNH2 (6) and the Aβ derived
host−guest switch peptide (Ac-SLSLHQKLVFF(H+)SEDVSL-
GNH2, 7) as the model aggregating peptides for our studies. O
to N acyl migration of these peptides was studied using LC−
MS (Figure 3a). Their aggregation potential was investigated
using various biophysical tools. In the CD spectrum, we
observed a band corresponding to β-sheet at pH 7.0 (Figure
3b), and further these peptides produced amyloid-like fibrils
Characterization of Aggregation Properties of Model
Aggregating Peptides. Mutter et al. reported -(Ser-Leu)n-
based switch-peptides that form amyloid-like fibrils rich in β
sheet in physiological pH and temperature.15 In these peptides,
one serine switch element was incorporated into a self-
aggregating peptide via depsi bond to control the kinetics of
self-assembly by adjusting the pH of the system. The switch
element present in the sequence helped the model aggregating
peptides to adopt a random coil conformation at acidic pH, as
the peptide fragments attached to it were shorter for β-sheet
formation for those sequences. When pH was adjusted from
neutral to basic range, O to N acyl migration occurred, resulting
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Figure 5. (a) Monitoring the intramolecular cyclization of peptide 4 by LC-MS at pH 7.4. (b) FT-IR spectrum of Aβ1−40 in the absence (black) and
presence (red) of peptide 4, checked after 4 days of incubation. (c) Mixing experiments of the Aβ peptide and the breaker peptides by CD at pH 7.4,
checked after 4 days of incubation. (d) Time dependent thioflavin T fluorescence studies of Aβ1−40 in the absence (black) and presence of peptide 4
(green) or 5 (red) at pH 7.4. (e) TEM micrographs of Aβ1−40 in the absence (upper panel) and presence (lower panel) of peptide 4, checked after 4
days of incubation. (f) Congo red birefringence image of Aβ1−40 in the absence (left) and in the presence (right) of peptide 4, checked after 4 days of
incubation. All studies pertaining to Aβ(1−40) were carried out at pH 7.4.
which was confirmed by TEM (Figure 3c). Also, we noticed a
characteristic green-gold birefringence from 4 day old peptide
solution upon staining with Congo red under polarized light
using a polarizable microscope (see the Supporting Informa-
tion, Figures S37 and S38) confirming amyloid formation.
Disruption of Fibril Formation of Model Amyloid
Forming Peptides by BBDP Containing Model Breaker
Peptides. We then set out to test the ability of the BBDP-
containing peptides to disrupt the aggregation of these model
aggregating peptides that were used as an Aβ mimic in this
study. Incremental fibril accumulation of the model aggregating
peptide 6 was observed up to 48 h in a thioflavin T fluorescence
study in the absence of the BBDP-containing breaker peptide 1
at pH 7.0. On the other hand, when peptide 1 was coincubated
with peptide 6 under similar experimental conditions, we
observed fibril formation up to only 6 h, which was slowly
disrupted thereafter (Figure 4a). Similarly, in a parallel
experiment, substantial decrease in the fluorescence signal of
peptide 7 was observed after 24 h when mixed with breaker
peptide 3 in 3-fold molar excess at pH 7.0 (Figure 4b). In
concordance with thioflavin T fluorescence, disruption of
amyloid was observed from TEM (see the Supporting
Information, Figures S33 and S34) and Congo red
birefringence studies (see the Supporting Information, Figures
S37c and S38c) as well. These findings support our hypothesis
that BBDP-containing peptide first aligns and coaggregates with
the neighboring aggregating peptide even at pH 7.0 when
sequence homology is maintained and as long as the breaker
element is not formed. Over time, at that pH, the breaker
element is generated in the form of a kink on the backbone due
to aspartimide formation. That kink slowly disrupts the self-
assembly as well as the aggregation of the neighboring peptide.
Ultimately, the ring-opening of the aspartimide, formation of
the α- and β-aspartyl peptide, and racemisation of the Asp
residue cause total collapse of the β-sheet architecture. In a
control experiment (Figure 4c), proline containing peptide
SLSPSLSGNH2 (8) inhibited the aggregation of peptide 6 from
the beginning, but the BBDP containing peptide SLSD(OBzl)-
SLSGNH2 (1) partially coaggregated and then disrupted. On
the other hand, mixing experiment with another control
peptide 9 (sequence: SLSDSLSGNH2), where D(OBzl) was
replaced with an unprotected Asp, shows extended lag time and
reduced amount of fibril, indicating it as a weak β sheet breaker.
Disruption of Fibril Formation of Aβ(1−40) by a BBDP
Containing Peptide. Finally, we designed and synthesized a
five membered BBDP-containing β-sheet breaker peptide (4),
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checked for chemical conversions (Figure 5a), and tested its
containing breaker peptide reaching to the target, it may work
as known β-breaker peptides.
efficiency for inhibition of aggregation of the full length Aβ1−40
.
It was designed to get a comparison with peptide 5, which is a
known β-sheet breaker peptide, and therefore, high sequence
homology was maintained with it. Aβ1−40 produced a sharp
band at 1631 cm−1 which was assigned to the amide I band of a
peptide in β-sheet conformation in FT-IR studies in the
absence of peptide 4. On the contrary, in the presence of
peptide 4, a new band emerged at 1646 cm−1 corresponding to
random coil (Figure 5b).
The influence of peptide 4 on the conformational change of
Aβ1−40 was also evident from CD studies (Figure 5c). Pure Aβ
peptide shows a characteristic curve of mixture of α-helix and β-
sheet (black curve). The mixture Aβ and peptide 5 show a
mixture of β-sheet and random coil (green curve). Whereas the
red curve of Figure 5c, which was obtained from the mixture of
Aβ and peptide 4, clearly indicates induction of a turn. Similar
results were obtained independent of amount of the breaker
peptides present in the solution (for 3-fold molar excess, Figure
5c; 5-fold, Supporting Information Figure S18; and 10-fold,
Supporting Information Figure S19).
A notable decrease in the thioflavin T fluorescence signal was
observed when Aβ1−40 was coincubated with peptide 5 as a
control experiment. Further decrement in the fluorescence
signal was observed when coincubated with similar excess of
peptide 4 (Figure 5d for 8-fold; see Supporting Information
Figures S28−S30 for 3-, 5-, and 10-fold molar excess of breaker
peptides). It was assumed that peptide 4 will form β-sheet at
the initial stage as long as the chemical conversion does not
take place. Templating effect of the Aβ peptide also may play its
role for coaggregation. The coaggregation was evident from the
increase in amount of fibril at the initial stage (until ∼24 h)
when Aβ was mixed with peptide 4 (Figure 5d, red curve).
Further decrease in the fluorescence signal was due to the
breaking effect after the chemical conversion. The chemical
conversion or intramolecular cyclization for peptide 4 took 24−
72 h at pH 7.4., which is in concordance with the results
obtained in fluorescence measurements.
CONCLUSION
■
We have demonstrated that peptides containing a BBDP unit
form a new generation of conformational switch peptide, which
forms β-sheet and fibril at the beginning and then breaks the
fibrillar nanostructure at a specific condition. It also easily aligns
with neighbor aggregating peptides due to the presence of
sequence homology and absence of the breaker element at the
initial stage easily, which then undergo a chemical conversion to
aspartimide-containing peptides at physiological pH and
generate a kink in the peptide backbone in situ. Formation of
aspartimide, subsequent ring-opening and racemisation of Asp
finally disrupt the native backbone of the breaker peptide. Such
chemical and structural changes of the BBDP unit forcefully
disrupt self-aggregation as well as aggregation of the
neighboring peptides upon coincubation, although the
aggregated state is thermodynamically most stable. Based on
all these findings, we conclude that the BBDP concept is valid
for β-sheet inhibition and disruption and may be useful for the
development of novel therapeutics against many amyloidoses
including Alzheimer’s disease. However, modulation of the
kinetics of the in situ intramolecular cyclization, reducing
toxicity caused by the leaving group, investigation on the
presence of the inherent toxicity of the designed molecules,
increasing ability to cross blood-brain barrier are some of the
remaining problems to be addressed. Nevertheless, many
compounds designed based on this concept may be used to
study formation and disintegration of aggregation at least in
vitro and may find biomedical applications for diverse
amyloidoses.
Furthermore, such a concept can also be useful for designing
novel material for biomedical application. For example, some
switchable hydrogel based drug carrier can be prepared using
BBDP concept, which may disrupt its architecture and release
the drug under physiological temperature and pH at the site.
We looked for fibril by TEM after 4 days of incubation.
Aβ1−40 alone produced fibrillar aggregates. However, amor-
phous aggregates were observed instead when coincubated with
peptide 4 for the same duration (Figure 5e). In agreement with
the TEM studies, a characteristic green gold birefringence was
noticed under polarized light from the Aβ1−40 sample and no
such birefringence was observed from the sample coincubated
with peptide 4, checked after 4 days (Figure 5f). This confirms
complete disruption of Aβ amyloid by BBDP-containing
breaker peptide (4). Importantly, there was no evidence of
reaggregation of the rearranged BBDP containing peptides.
Some of the results (for example, comparison of the red
curve and green curve in Figure 5d and comparison of the red
curve and blue curve in Figure 4c) suggest that β-breaking
efficiency of BBDP containing peptide (peptide 4) is
comparable to that of the preinstalled breaker element
containing peptide homologue (peptide 5), if not better.
Nevertheless, the problem of binding to the target peptides due
to the preinstalled breaker element can be bypassed using such
a prodrug type concept. However, it is necessary to modulate
the kinetics of mentioned chemical conversion, which may be
achieved by varying the side chain protection of Asp of BBDP,
so that it takes place at physiological pH but not before it
reaches to the target aggregating peptide, soluble oligomers, or
amyloid in vivo. If the conversion occurs before the BBDP
METHODS
■
Peptide Synthesis. Peptides were synthesized by stepwise solid
phase peptide synthesis using Fmoc/tBu protection strategy on Rink
amide resin (loading 1.1. mmol/g). Resin was swollen in DCM for 1.5
h, and DMF for 0.5 h. Two equivalents of Fmoc amino acids, 2.5 equiv
of PyBOP, and 5 equiv of DIPEA were used. Coupling time varied
from 45 min to 6 h. Each coupling was monitored by Kaiser test, and
in the case of incomplete acylations coupling cycle was repeated
followed by capping. Fmoc removal was achieved using 20% piperidine
in DMF mixture for 21 min (7 min × 3). After final Fmoc removal,
peptide was cleaved from the resin using TFA/DCM/H2O (8:1.5:0.5)
for 3 h. Crude peptide was precipitated by cold diethyl ether and then
centrifuged.
Crude peptides were purified on a Waters 600E semipreparative
RP-HPLC using a C18-μ Bondapak column at a flow rate of 5 mL/
min. The solvent system was composed of solvent A (0.09% TFA in
H2O) and solvent B (0.09% TFA in CH3CN). A Waters 2489 UV
detector was used with an option of dual detection at 214 and 254 nm.
Gradient used for purification was 5−20% CH3CN for 5 min, 20−60%
CH3CN over 5−25 min.
Aβ Sample Preparation.16 An amount of 0.5 mg of commercially
available Aβ(1−40) was dissolved in TFA to obtain homogeneous
solution free of aggregates. TFA was evaporated using N2. To remove
TFA completely, HFIP was added and evaporated using N2 to obtain a
filmlike material. This process was repeated twice. A volume of 2.0 mL
of PBS (50 mM, pH 7.4) was added to the Eppendorf tube followed
by sonication and vortexing to obtain a final concentration of 50 μM.
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Research Article
Specified volume from this stock solution was used for various
experiments.
Fourier Transform Infrared Spectroscopy. A volume of 20 μL
of the peptide sample after 4 days of incubation was mixed with KBr,
and a pellet was prepared. To obtain final spectra, the background scan
was subtracted from the sample scans and text files were plotted using
Origin 61 software.
and Dr. H. Clark Hyde and Dr. K. Mandal for language
correction.
REFERENCES
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ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and copies of spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
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AUTHOR INFORMATION
Corresponding Author
*E-mail: bmandal@iitg.ernet.in.
■
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Author Contributions
K.C.N. and A.P. synthesized the compounds and performed the
experiments. K.C.N. and B.M. designed the experiments and
wrote the paper. All authors approved the submission.
Funding
We are thankful to BRNS-DAE for financial support (Young
scientist research award grant, Sanction No. 2008/20/37/6/
BRNS).
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We are thankful to CIF, IIT Guwahati for LC-MS and TEM
studies; Mr. Sameer Hussain and Prof. P. K. Iyer for
birefringence studies; DBT program facility for CD studies;
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Research Article
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