Amyloid peptides incorporating a core sequence from the amyloid beta peptide and gamma amino acids: Relating bioactivity to self-assembly

Article abstract of DOI:10.1039/c1cc15493a

A series of heptapeptides comprising the core sequence Aβ(16-20), KLVFF, of the amyloid β peptide coupled with paired N-terminal γ-amino acids are investigated in terms of cytotoxicity reduction and binding to the full Aβ peptide, both pointing to inhibition of fibrillisation for selected compounds. This is related to the self-assembly capacity of the heptapeptides.

Full text of DOI:10.1039/c1cc15493a

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COMMUNICATION
Amyloid peptides incorporating a core sequence from the amyloid beta
peptide and gamma amino acids: relating bioactivity to self-assemblyw
Valeria Castelletto, Ge Chengz and Ian W. Hamley*
Received 5th September 2011, Accepted 19th October 2011
DOI: 10.1039/c1cc15493a
A
series of heptapeptides comprising the core sequence
affect fibrillisation.18 In related work, SREs have been developed
based on core sequences of the amyloid-forming peptide
a-synuclein, modified using b-alanine or g-aminobutyric acid
(GABA).19 Reduced aggregation of a-synuclein in the presence
of SREs was observed, and one of the b-alanine modified peptides
studied also inhibited aggregation of Ab(1–40). Non-natural
amino acids such as b- and g-amino acids are of interest since
they are resistant to proteolysis, enabling the preparation of
compounds designed to incorporate biostable motifs.
Ab(16–20), KLVFF, of the amyloid b peptide coupled with
paired N-terminal c-amino acids are investigated in terms of
cytotoxicity reduction and binding to the full Ab peptide, both
pointing to inhibition of fibrillisation for selected compounds.
This is related to the self-assembly capacity of the heptapeptides.
Intense research activity is focussed on the development of
treatments for Alzheimer’s disease. The aggregation of the
amyloid b peptide into oligomers or fibrils is now implicated as
a key process associated with progression of this disease.1–4
Several mechanisms for cytotoxicity have been suggested,2,5,6
of which the most well known is probably the Ab-induced
formation of ion channels in cell membranes. A number of
strategies are actively being pursued by research teams in academia
and the pharmaceutical industry.7–9 These include (i) development
of g-secretase inhibitors (g-secretase is an enzyme involved in
cleavage of amyloid b (Ab) peptides from the Amyloid Precursor
Protein), (ii) passive immunization based on Ab antibodies
(iii) inhibition of aggregation of oligomers.
We investigated a series of g-amino acid peptides containing
the KLVFF sequence or its D-amino acid variant (Scheme 1)
in cell viability studies using the MTT assay with SH-SY5Y
neuronal cells. It should be noted that compound II is
bAbAKLVFF, the subject of a previous study.18
We investigated the effect of compounds I–V on the viability
of SH-SY5Y cells using an MTT reduction colorimetric assay.
Dose response curves are presented in Fig. 1. These results
show that Ab(1–42) leads to a loss of cell viability to around
30%. The g-amino acid containing peptides alone do not cause
cell death, cell viability remains above 90%. Unfortunately,
I and II do not reduce the cytotoxicity of Ab(1–42) (this
conclusion for II is in agreement with our previous report).18
However, compounds III, IV and V show a substantial
Here, we report on the development of a series of novel
compounds that show promising in vitro properties in binding
to Ab and in restoring viability of neuronal cells. Inhibition of
oligomer aggregation has been targeted via use of self-recognition
elements (SREs); these are molecules based on fragments of the
Ab peptide which are capable of binding to the corresponding
sequence, but modified so as to disrupt b-sheet fibrillisation.
Findeis et al. showed that compounds based on a core sequence
of the Ab peptide implicated in fibrillisation, Ab(16–20),
KLVFF, showed promise as SREs.10 Other groups have
developed related approaches based on extended or modified
KLVFF sequences11–15 or oligomers with complementary
residues16 or non-peptidic compounds designed to recognise
KLVFF.17 We have recently investigated the interaction of
bAbAKLVFF (bA = b2-alanine) with Ab(1–42) and showed
that the bA-modified heptapeptide can bind Ab(1–42) and
Dept of Chemistry, University of Reading, Whiteknights, Reading,
RG6 6AD, UK. E-mail: I.W.Hamley@reading.ac.uk
w Electronic supplementary information (ESI) available: Experimental
Methods, results from CD and ThT binding studies, XRD patterns
and spacings, additional FTIR spectra and images of hydrogels. See
DOI: 10.1039/c1cc15493a
Scheme 1 Peptides studied. I, III, IV, V: N-terminal g-amino acid
modified KLVFF SRE peptides. Compound II contains N-terminal bA
residues, this compound, studied previously by us,18 is included as a
reference sample. It may be noted that in compounds III, IV and V, the
KLVFF sequence is based on D-amino acids. In the following, the influence
of these non-natural a-amino acid residues on self-assembly is examined.
z Present address: Dept of Chemistry, University of Liverpool, Crown
Street, Liverpool L69 3BX, UK.
c
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Fig. 2 Cryo-TEM images for peptides I (3 wt%), III (2 wt%) and IV
Fig. 1 Dose-response curves based on MTT cell viability assay for
selected compounds in the presence or absence of Ab(1–42).
(5 wt%) and V (3 wt%). The scale bars represent 200 nm.
of b-sheets, since a characteristic ‘‘cross-beta’’ pattern24 is
observed (ESIw Fig. 4) with strong meridional reflections from
the spacing of beta strands aligned perpendicular to the
vertical fibril axis, along with a series of equatorial reflections
from spacings within and between b-sheets.
dose-dependent increase in cell viability, in the case of peptide
III to around 90%, at physiologically relevant concentrations
of Ab(1–42) and peptide.
To investigate the binding of our compounds to Ab(1–42) in
more detail, circular dichroism (CD) spectroscopy experi-
ments were performed. CD also provides information on the
self-assembly of the peptides in the absence of Ab(1–42). The
resulting data are presented in ESIw Fig. 1 (measured spectra)
and ESIw Fig. 2 which shows the measured spectra for
mixtures of Ab (50 mM) with I, II, IV and V at the molar
ratios shown, along with calculated spectra based on addition
of the measured spectra for the two components. These spectra
reveal that compounds III and V show the strongest binding
characteristics, in fact even at the lower molar ratio of added
heptapeptide studied the measured curves deviate significantly
from those calculated. At higher molar ratios, the CD spectra
for these systems become rather flat pointing to loss of
secondary structure upon binding of peptide to Ab(1–42).
The finding that III in particular binds strongly to Ab and
disrupts b-sheet structure correlates to the cytotoxicity behaviour
shown in Fig. 1. Dye binding studies using thioflavin T (ESIw
Fig. 3) also show binding of III to Ab(1–42).
It may be noted that the CD spectrum for III (ESIw Fig. 1)
shows significantly stronger features (in particular a maximum
at 225 nm) than the other peptides. This suggests that the reduced
cytotoxicity when adding III to Ab(1–42) and increased binding
of these species, may be related to the intrinsic self-assembly
properties of III. These were therefore investigated in more
detail, in comparison with the other peptides. The self-assembly
of g-amino acid based peptides is also attracting considerable
attention at present in its own right due, for instance, to
enhanced properties such as resistance to proteolysis,20 formation
of unique secondary structures20–22 and also the ability to mimic
natural secondary structures using non-natural amino acids.23 In
contrast to previous work, the incorporation of a b-sheet forming
a-amino acid motif (KLVFF) along with two g-amino acid
residues in I, III, IV and V inhibits the formation of the more
common helical structure of g-amino acid peptides, and instead
amyloid b-sheet structures are observed.
A table listing observed reflections is provided as ESIw
Table 1. An interesting feature was noted for all samples
except II, the subject of previous papers25,26 and III, in that
the main meridional reflection was split into a doublet, with
the strongest peak corresponding to a spacing d = 4.94 A along
with a lower intensity peak from a spacing d = 4.73–4.77 A.
This latter spacing is in the range usually associated with the
inter-strand distance in a b-sheet,27,28 however the increased
spacing d = 4.94 A also observed for these peptides points to an
expansion of the inter-strand distance and is ascribed to folding
of the g-amino acid residues at the N terminus. This hypothesis
is supported by the fact that the spacing associated with the
length of the peptide (ESIw Table 1) is significantly smaller than
that calculated for an a-amino acid heptapeptide.29
FTIR and CD were used to investigate the secondary structure of
the peptides, as a function of concentration (at higher concentration
than the CD data in ESIw Fig. 1 and ESIw Fig. 2). Fig. 3 shows
CD and FTIR spectra measured in in two concentration
ranges. The FTIR spectra in the amide I0 region in more dilute
solution (Fig. 3a) show that only III forms b-sheets in dilute
solution since for this sample there is a peak at 1618 cmꢀ1 (with a
shoulder at 1626 cmꢀ1). All spectra also show a peak at 1672 cmꢀ1
Fibrillisation of these peptides was directly confirmed by
cryogenic-TEM. Fig. 2 shows representative images. The fibril
diameter for all four peptides is approximately 10 nm.
X-Ray diffraction from stalks prepared from 1–1.8 wt%
solutions (except for I for which stalks could not be prepared
unless the concentration was increased) confirmed the presence
Fig. 3 FTIR and CD spectra. (a, b) FTIR and CD spectra for
0.4 wt% solutions, (c,d) FTIR and CD spectra for 1 wt% solutions.
c
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Chem. Commun., 2011, 47, 12470–12472 12471

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due to TFA counterions.27 For III, the absence of a peak
around 1680 cmꢀ1 indicates that the b-sheets have a parallel
configuration. Samples I and IV show a peak at 1640 cmꢀ1
which is characteristic of either random coil or polyproline II
structure (as discussed elsewhere,30 the two are difficult to
distinguish based on standard FTIR spectroscopy alone).
Sample V shows mainly the 1640 cmꢀ1 peak, with a shoulder
indicating some contribution from b-sheet structures.
not observed for III, possibly contributing to the enhanced
fibrillisation properties of this peptide. These findings provide
insight into the self-assembly of this novel class of peptides.
The combination of g-amino acids with an Ab SRE motif
(KLVFF) provides a new class of fibrilisation inhibitor.
Notes and references
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The FTIR spectra assist in assignment of the CD spectra
(Fig. 3b), which are often difficult to interpret for short
peptides that do not exhibit classical a-helical or b-sheet
spectra. Consistent with the FTIR data, III shows a distinct
spectrum with a maximum just above 200 nm and a minimum
at 213 nm as expected for b-sheets. The spectra for all samples
(except I) also contain a peak or shoulder at 230 nm which is
ascribed to phenylalanine stacking interactions.31 The spectra
suggest random coil rather than PPII structure is predominant.
Turning to Fig. 3c,d, when concentration is increased to 1 wt%,
FTIR shows clear features of (parallel) b-sheets for all peptides
except I which retains a random coil conformation. Interestingly,
the CD data for samples at 1 wt% do not show clear b-sheet like
features, i.e. a minimum in the range 215–220 nm. We believe
that contributions from aromatic residues at the C and N termini
of the spectra predominate in these CD spectra.31–33 At still
higher concentration (2–3 wt%, comparable to that used in the
cryo-TEM studies), FTIR confirmed the presence of features
associated with b-sheets in the amide I0 region for all samples
(ESIw Fig. 5). A further interesting observation for several of
these compounds was the formation of self-supporting hydrogels
at sufficiently high concentration. ESIw Fig. 6 shows photographs
of representative systems. Formation of these hydrogels is
associated with b-sheet features in the FTIR spectra and suggests
they comprise a fibrillar network structure. These hydrogels
might be useful in the development of slow release systems.
In summary, we have discovered a promising lead compound
(III) containing two g-amino acid residues [(R)-(4-amino-
5-phenyl]pentanoyl and the D-amino acid version of KLVFF
(sometimes denoted klvff).10 This material shows a favorable
dose-response curve, reducing the cytotoxicity of Ab in a
physiologically relevant concentration range. In turn we show
by CD spectroscopic and ThT dye fluorescence studies that this
peptide binds to Ab. This led us to examine whether the
bioactivity of this peptide could be related to its self-assembly
propensity. Indeed, we find that peptide III forms b-sheet
structures at lower concentration than the other samples
examined. Another unanticipated finding was the splitting of
the b-strand spacing in the fibre XRD patterns of several of
the peptides, a feature we associate with the inability of the
N-terminal g-amino acid residues to fit into the regular
H-bonding pattern of the a-amino acid residues. This was
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12472 Chem. Commun., 2011, 47, 12470–12472
This journal is The Royal Society of Chemistry 2011