Peptidomimetics That Inhibit and Partially Reverse the Aggregation of Aβ1-42

Article abstract of DOI:10.1021/acs.biochem.7b00223

The peptide sequence KLVFF resembles the hydrophobic core of the Aβ peptide known to form amyloid plaques in Alzheimer's disease. Starting from its retro-inverso peptide, we have synthesized three generations of peptidomimetics. Step by step natural amino acids have been replaced by aromatic building blocks accessible from the Pd-catalyzed Catellani reaction. The final compound 18 is stable against proteolytic decay and largely prevents the aggregation of Aβ_1-42 over extended periods of time. The activity of the new inhibitors was tested first by fluorescence correlation spectroscopy. For closer examination of compound 18, additional techniques were also applied: laser-induced liquid bead ion desorption mass spectrometry, confocal laser scanning microscopy, thioflavin T fluorescence, and gel electrophoresis. Compound 18 not only retards the aggregation of chemically synthesized Aβ but also can partially dissolve the oligomeric structures. Thioflavin binding mature fibrils, however, seem to resist the inhibitor.

Full text of DOI:10.1021/acs.biochem.7b00223

Article
pubs.acs.org/biochemistry
Peptidomimetics That Inhibit and Partially Reverse the Aggregation
of Aβ1−42
Tina Stark,† Tobias Lieblein,‡ Maximilian Pohland,§ Elisabeth Kalden,† Petra Freund,‡ Rene Zangl,‡
́
Rekha Grewal,∥ Mike Heilemann,‡ Gunter P. Eckert,§,⊥ Nina Morgner,‡ and Michael W. Gobel*,†
̈
‡
§
†Institute of Organic Chemistry and Chemical Biology, Institute of Physical and Theoretical Chemistry, and Department of
Pharmacology, Goethe University Frankfurt, Campus Riedberg, Max-von-Laue-Strasse 7-9, D-60438 Frankfurt am Main, Germany
∥Institute of Nutritional Sciences, Justus-Liebig-University Giessen, Wilhelmstrasse 20, D-35392 Giessen, Germany
S
* Supporting Information
ABSTRACT: The peptide sequence KLVFF resembles the hydrophobic core of the
Aβ peptide known to form amyloid plaques in Alzheimer’s disease. Starting from its
retro-inverso peptide, we have synthesized three generations of peptidomimetics. Step
by step natural amino acids have been replaced by aromatic building blocks accessible
from the Pd-catalyzed Catellani reaction. The final compound 18 is stable against
proteolytic decay and largely prevents the aggregation of Aβ1−42 over extended periods
of time. The activity of the new inhibitors was tested first by fluorescence correlation
spectroscopy. For closer examination of compound 18, additional techniques were also
applied: laser-induced liquid bead ion desorption mass spectrometry, confocal laser scanning microscopy, thioflavin T
fluorescence, and gel electrophoresis. Compound 18 not only retards the aggregation of chemically synthesized Aβ but also can
partially dissolve the oligomeric structures. Thioflavin binding mature fibrils, however, seem to resist the inhibitor.
lzheimer’s disease (AD) is the most common cause of
peptide to RI-OR2 led to an analogue (RI-OR2-TAT) that
Adementia affecting around 47 million people worldwide in was able to cross the blood−brain barrier and to reduce the
2015.1,2 Despite the long search for therapies, still no cure for
plaque load, the level of oxidative damage, and the extent of
inflammation in a transgenic mouse model of Alzheimer’s
AD exists. Thus, developing concepts to counteract this illness
is of great relevance.3−6 Pathologically, amyloid plaques
composed of the Aβ peptide and neurofibrillary tangles of
hyperphosphorylated Tau protein are found in the brains of AD
patients. The Aβ peptide is formed from the amyloid precursor
protein (APP) by proteolytic cleavage through β- and γ-
secretase. Because of different cleavage sites, various Aβ
isoforms 38−43 amino acids in length exist, with Aβ1−40 and
Aβ1−42 being the most common. According to the amyloid
hypothesis, the aggregation of Aβ initiates a complex cascade
that ultimately leads to neuronal cell death and loss of brain
functions.7,8 Two recent studies by solid-phase nuclear
magnetic resonance (NMR) have elucidated the three-dimen-
disease.17
Other inhibitors derived from the KLVFF sequence contain
N-methylated amino acids,18,19 α,α-disubstituted amino
acids,20,21 or alternating amide and ester bonds.22 It is also
possible to combine several KLVFF sequences into a single
molecule.23,24 Apart from KLVFF derivatives, β-sheet breaker
peptides are quite prominent as Aβ aggregation inhibitors.
These peptides are homologous to Aβ and likewise hydro-
phobic but have a weakened tendency to form β-sheets.25 One
example is the peptide LPFFD26 and modifications thereof.27,28
The central hydrophobic part of Aβ is important for
aggregation, as is the C-terminus. Peptides corresponding to
this sequence were also tested as inhibitors.29−31 Examples of
nonpeptidic aggregation inhibitors are molecular tweezers,32,33
aminopyrazoles,34,35 scyllo-inositol,36 tramiprosate,37 and poly-
phenols such as curcumin, resveratrol, and epigallocatechingal-
late.38
sional structure of amyloid fibrils formed by Aβ1−42.
9,10 It is well
documented, however, that, not the Aβ fibrils themselves, but
soluble oligomers of Aβ are the most cytotoxic agents.11,12
Thus, preventing the early phase of Aβ aggregation is a
promising concept for impeding the progress of AD.6,12
The aggregation of molecules may be analyzed by mass
spectrometry (MS) provided the species distribution in the gas
phase mirrors the situation of the liquid phase. This requires
the mildest possible ionization conditions.
It has been known for many years that the KLVFF sequence
(Aβ16−20) plays an important role in the aggregation of Aβ.13
Many peptides derived from this sequence inhibit the
aggregation process.14 Two examples are peptides OR1 and
OR2 (RGKLVFFGR), where arginine was added to the
hydrophobic core to enhance the solubility. The glycine units
serve as spacers.15 As these inhibitors suffer from instability
against proteases, a retro-inverso derivative (RI-OR2) was
synthesized, which is not degraded by proteases and retains its
inhibitory activity.16 Attachment of a retro-inverso TAT
Aβ aggregation has been analyzed previously by electrospray
ionization (ESI) mass spectrometry.39−42 Such experiments are
Received: March 10, 2017
Revised: July 28, 2017
© XXXX American Chemical Society
A
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challenging because of the fact that the mass spectra show
mainly the monomer and some smaller peaks at very low
intensities that can be assigned to the dimer and a few higher
Scheme 1. Synthesis of Amino Acid 2a and of Precursors
1a−h
a
species. This challenge is even more pronounced with Aβ1−42
,
the amyloid peptide most prone to aggregation. Therefore, it
was not possible to study the aggregation process over longer
periods of time because the magnitudes of the small oligomer
signals decreased further because of the formation of larger
oligomers and fibrils.41 Nonetheless, the effect of inhibitor
molecules on the aggregation of Aβ1−42 has been investigated
by ESI.43−45
Recently, we established LILBID-MS, a technique that can
detect weak molecular complexes, as a new tool to follow the
aggregation kinetics of Aβ.31 Oligomers up to 20-mer as well as
Aβ−inhibitor complexes can be observed and quantified. Larger
aggregates were detected in this study by simultaneous
application of fluorescence correlation spectroscopy (FCS)
and transmission electron microscopy (TEM). Furthermore,
we could show that these methods can be used to identify
inhibitors of aggregation. Whereas FCS and TEM are sensitive
to only large oligomers and fibrils, LILBID clearly demonstrates
if an inhibitor can also stop the first steps of oligomerization.
The best results were found for the KLVFF derivative OR2.31
In the study presented here, we modified its retro-inverso
peptide in three steps by replacing hydrophobic amino acids
with non-natural building blocks that can be accessed from the
Catellani reaction.46,47 The final compound 18, stable against
proteolytic degradation, retards the aggregation of chemically
synthesized Aβ1−42 over 7 days at 37 °C and also partially
dissolves pre-aggregated samples.
a
(a) (1) KHMDS, dry THF, −78 °C; (2) trisyl-azide, dry THF, −78
°C; (3) HOAc, 30 °C; (b) H2O2, LiOH, THF/H2O, 0 °C; (c) Pd/C,
H2, MeOH, room temperature (ref 47); (d) Boc2O, NaHCO3,
dioxane/H2O, 0 °C, room temperature.
a
Scheme 2. Synthesis of Peptidomimetic 5f
RESULTS
■
The Catellani reaction is a Pd-catalyzed multicomponent
reaction.46 It may be used to attach two or three side chains
to an aromatic framework in a single synthetic step. Products
can be converted stereoselectively into α-amino acids equipped
with a second amino alkyl chain as shown for the 1h → 2a
example (Scheme 1). In the same way, we have prepared α-
amino acids from intermediates 1b−g.47 However, in the work
presented here, with exception of compound 2a, the α-amino
residue was not required. Hence, compounds 1b−g were
hydrolyzed to form the achiral Boc-protected amino acids 3b−
h (Scheme 2 and the Supporting Information).
a
(a) H2O2, LiOH, THF/H2O, 0 °C; (b) D-Val-OMe·HCl, DCC,
HOBt, DIPEA, CH2Cl2, room temperature; (c) NaOH, MeOH/H2O,
room temperature; (d) D-Lys-D-Leu-NH2 (resin-bound), DIC, HOBt,
NMP, room temperature; (e) TFA/TIS/H2O, room temperature;
HPLC purification. For all other compounds 3 and 4, see Scheme 1 for
the aromatic residue.
In the first generation, the two N-terminal Phe residues of
retro-inverso KLVFF were replaced by one of the non-natural
amino acids (2a or 3b−h) to generate compounds 5a−h
(Chart 1).
Scheme 2 shows exemplarily how compound 5f was
synthesized by a combination of solution- and solid-phase
methods. Carboxylic acid 3f reacted first with D-valine methyl
ester in solution. After saponification, this fragment was
coupled to the D-Leu-D-Lys part, which had been assembled
before on a solid support (Scheme 2).
To evaluate compounds 5a−h, we used an assay based on
FCS that is well suited for a fast and easy identification of
inhibitors. Aβ (100 μM) and a Cy5-labeled analogue of this
peptide (0.5 μM) are incubated at 4 °C in the presence of the
potential inhibitor (400 μM).31 After different periods of time,
samples are 10-fold diluted and analyzed by FCS. The Cy5-
labeled Aβ is integrated into the Aβ oligomers and fibrils, so
that these can be detected by FCS. With the increasing size of
aggregates, increasing diffusion times are expected. The
instrument’s software allows the autocorrelation function to
be formally fitted to a one-, two-, or three-component system.
This is a crude approximation, as aggregating Aβ samples are
composed of large numbers of individual oligomers. Never-
theless, the mixture can be roughly categorized as monomers
and small oligomers (diffusion time of 80 μs), medium-sized
oligomers (diffusion time of ∼5000 μs), and large aggregates
(diffusion time of >50000 μs). We have shown that the fraction
with the shortest diffusion time of ∼80 μs represents mainly
monomeric Aβ with a few small oligomers (hereafter named
“monomeric Aβ” for the sake of simplicity). During the
aggregation of an Aβ sample, the fraction of monomeric Aβ
decreases, while the amount of medium- and large-sized
aggregates increases. Therefore, if a molecule inhibits
aggregation, the percentage of the monomeric fraction will
remain near 100% over the whole incubation time.31
Within the first generation of peptidomimetics, those
containing small aromatic systems (5b−d) showed the fastest
decay of the monomer fraction (Table 1). Stronger inhibition
was seen with CF3 analogue 5e. The best structures were those
B
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Chart 1. Inhibitors of the First Generation Compared to OR1, OR2, and the Retro-Inverso Peptide of KLVFF
a
Table 1. Inhibition of the Aβ Aggregation by 5a−h Measured with FCS
b
incubation time (h)
0
6
24
72
192
20
+48
0
c
control
93
79
94
92
36
26
d
e
5a
94
8
7
3
6
100
0
100
0
1
96
5
4
97
0
9
d
h
h
h
5b
100
95
0
83 12
60 18
70 11
nd
nd
nd
d
h
h
h
5c
78 20
81 11
nd
nd
nd
d
h
h
h
5d
100
100
97
0
0
nd
nd
nd
d
h
h
5e
94
96
3
3
87
91
97
88
96
1
2
2
2
5
96
6
nd
nd
d
5f
2
100
99
100
97
0
91
d
5g
100
100
100
0
0
0
86 14
1
0
89 10
e
h
h
5h
70
7
91
nd
nd
d
,
f
g
h
h
h
OR2
100
0
nd
nd
nd
a
The percentage of the fastest fraction (diffusion time of ∼80 μs) is shown, corresponding mainly to Aβ monomers. Samples were incubated at 4 °C
b
c
d
e
with a 4-fold excess of inhibitor. Further incubation at 37 °C, after 192 h at 4 °C. Single experiment. Mean value of three experiments. Mean
value of two experiments. Data taken from ref 31. Measured after 4 h at 4 °C. Not determined.
f
g
h
with condensed aromatic systems: naphthalene 5f, phenan-
threne 5g, and pyrenes 5a and 5h. Therefore, the incubation
time of such samples was extended to 8 days at 4 °C (192 h),
when the samples still consisted mainly of monomeric Aβ
species. After additional incubation at 37 °C for 2 days, the
monomeric fraction completely disappeared in the control. In
contrast, samples with inhibitors 5a, 5f, and 5g still contained
high levels of monomeric Aβ. These results also demonstrate
that the α-amino group present in compound 5a may be helpful
but is no requirement for good inhibitory activity.
the C-terminal carboxamide. Pyrene analogue 13h was
prepared in the same way.
In the FCS assay, naphthalene 13f showed activity (89 4%
monomeric Aβ) that was higher than that of pyrene 13h (73
1% monomeric Aβ) after incubation for 24 h at 4 °C. This
became even more pronounced after subsequent incubation at
37 °C for 3 days. Whereas samples containing 13f still mainly
consisted of monomeric Aβ (89 3%), samples containing 13h
showed an obvious decrease in the monomer fraction (49
9%).
The promising effects of compounds 5a−h prompted us to
further simplify the structures of the inhibitors. Scheme 3 shows
the synthesis of compound 13f, derived from 5f by omission of
In compounds 5a−h, 13f, and 13h, the FF part of the
KLVFF sequence and its retro-inverso counterpart has been
mimicked by the different aromatic ring systems, each equipped
C
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a
Scheme 3. Synthesis of Peptidomimetic 13f
a
(a) Boc2O, Et3N, THF, 0 °C; (b) Cbz-D-Leu-OH, HATU, DIPEA, DMF, room temperature; (c) H2, Pd/C, THF, room temperature; (d) H2, Pd/
C, MeOH, room temperature; (e) (1) NaOH, MeOH/H2O, room temperature; (2) D-Val-OMe·HCl, HATU, DIPEA, DMF, room temperature; (f)
(1) NaOH, MeOH/H2O, room temperature; (2) 9, HATU, DIPEA, DMF, room temperature; (g) TFA, CH2Cl2, room temperature; HPLC
purification (as TFA salt).
with a basic side chain. Our most promising candidate, 18,
belongs to the third generation of inhibitors. In this structure,
we also exchanged the KL part with a synthetic ring system. Its
amino group should replace the side chain of lysine, whereas
the aromatic residue was a hydrophobic substitute for leucine.
The synthesis started from methyl ester 10f. After hydro-
genation, the Boc protecting groups were replaced by Cbz. The
ester was then reduced and the resulting alcohol converted into
amine 16. The removal of Cbz was followed by coupling to
naphthyl-D-Val building block 11f. Global deprotection with
TFA resulted in molecule 18 (Scheme 4).
concentrations of 10 μM Aβ and 40 μM 18, precipitation
could no longer be observed. Initial FCS experiments in this
concentration range looked very promising. All subsequent
experiments, therefore, were focused on this candidate.
Three types of experiments have been conducted and
analyzed by four independent analytical methods: FCS, LILBID
mass spectrometry, thioflavin T (ThT) fluorescence, and
confocal laser scanning microscopy (CLSM). First, we have
studied the aggregation of Aβ in the absence of 18. In the next
set of experiments, the inhibitor was present from the
beginning on. In the third type of assays, compound 18 was
added to a pre-aggregated sample to see if oligomers and fibrils
of Aβ might be dissolved again.
In the first experiments, it turned out, however, that the
presence of two naphthalene units reduced the solubility of 18
in water and caused precipitation at 400 μM. A slight
adaptation of assay conditions was thus required. At
Aβ Aggregation in the Absence of Compound 18.
Even at Aβ concentrations of 10 μM, fast aggregation could be
observed with FCS (Table 2, control). As described previously,
a
a
Scheme 4. Synthesis of Peptidomimetic 18
Table 2. Summary of FCS Measurements
incubation time (days)
control
0
2
0
7
0
0 μM 18
100
0
0
0
b
inhibition
20 μM 18
40 μM 18
20 μM 18
40 μM 18
nd
70 16
88 16
44 30
75 24
81 26
99
65 30
63
b
nd
1
b
c
c
dissolution
nd
b
nd
5
a
The percentage of the fast-moving fraction (diffusion time of ∼80 μs)
is shown, corresponding mainly to Aβ monomers. Samples were
incubated at 37 °C. The mean values of three experiments are given.
b
c
Not determined. Measured after aggregation for 2 days and 1 h after
addition of 18. Initial concentrations: 10 μM Aβ and 0.05 μM Cy5-Aβ.
the exact kinetics of Aβ aggregation is difficult to reproduce.
Impurities from solid-phase synthesis that can hardly be
removed by HPLC are known to retard the process.48 On
the other hand, different samples may contain varying amounts
of oligomeric species even at time zero. Such oligomers serve as
nucleation sites, thereby enhancing the kinetics. This well-
known problem has led to the development of special protocols
for the preparation of recombinant Aβ by size-exclusion
a
(a) H2, Pd/C, MeOH, room temperature; (b) (1) TFA, CH2Cl2,
room temperature; (2) CbzCl, Na2CO3, THF/H2O, 0 °C, room
temperature; (c) LiBH4, dry THF, Δ; (d) (1) PPh3, I2, imidazole, dry
DCM, 0 °C; (2) HNBoc2, Cs2CO3, dry DMF, 90 °C; (e) (1) Pd/C,
H2, MeOH, room temperature; (2) 11f, NaOH, MeOH/H2O, room
temperature; (3) HATU, DIPEA, dry DMF, room temperature; (f)
TFA, CH2Cl2, room temperature; HPLC purification (as TFA salt).
D
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Figure 1. Comparison between high and low mass settings. LILBID spectra of an Aβ sample (100 μM) incubated at 4 °C in the absence of an
inhibitor. (A) Spectrum with high mass settings obtained directly after dissolving Aβ. (B) Spectrum of Aβ after incubation for 48 h using high mass
settings. The different Aβ oligomers are indicated. (C) Spectrum of the same freshly dissolved sample as in panel A recorded using low mass settings.
(D) Spectrum of the same incubated sample as in panel B using low mass settings.
Figure 2. LILBID spectra of Aβ (100 μM) with and without a 4-fold excess of compound 18. The sample was incubated for 48 h at 4 °C. The inlet
shows the monomer/oligomer ratios for these spectra.
chromatography. Samples resulting from this method aggregate
exceptionally fast with reproducible kinetics.6,49,50 In the study
presented here, however, chemically synthesized Aβ has been
used.
Furthermore, determining the aggregation kinetics by FCS is
difficult, as the sample is composed of so many different species
that a meaningful mathematical analysis is nearly impossible.
We therefore focus on the amount of the monomeric Aβ
fraction that will decrease over time but should stay constant if
aggregation is stopped by an inhibitor. After 2 days at 37 °C,
this monomeric fraction consistently dropped to zero in the
absence of 18 and the particle number declined by 1 order of
magnitude.
with individual concentrations that quickly fall below the
detection limit for LILBID. Therefore, all experiments had to
be performed at 100 μM and 4 °C as described previously.31
Panels A and B of Figure 1 depict Aβ directly after dissolution
as well as after aggregation for 48 h, showing clearly the first
oligomerization steps. Thus, LILBID can detect the early
species in the Aβ aggregation process as seen here for (Aβ)1−
(Aβ)17. Because the instrumental settings used for the analysis
of these higher-order oligomers are not optimal for the
investigation of the monomers and dimers, panels C and D of
Figure 1 show the same samples at different settings, which
improve the signal/noise ratio of the low-mass species. It is
obvious that using the low mass settings increases the
sensitivity for the monomers and dimers but still enables us
to monitor the oligomerization process up to the decamers.
Unfortunately, at Aβ concentrations of 10 μM, the
aggregation leads to a wide heterogeneity of oligomeric species
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Figure 3. LILBID spectra of an Aβ sample (100 μM) incubated for 48 h at 4 °C (black) and treated afterward with 400 μM inhibitor 18 for 70 min
(blue) and 48 h (red). The different Aβ oligomers are indicated. The inlet shows the monomer/oligomer ratio for those three spectra.
These settings have been used for the inhibition studies
(Figures 2 and 3), as we are interested specifically in the
distribution of monomers and small oligomers.
Via detection of the fluorescence of thioflavin T, which
intercalates into sheets of Aβ, fast aggregation was also
confirmed.51 After 2 days at 37 °C, the fluorescence reached
74% of the maximum value obtained after 7 days (see Table 3,
control).
ments, Aβ (10 μM) was incubated at 37 °C in the presence of a
2- or 4-fold molar excess of the inhibitor. Samples were then
examined after 2 and 7 days. The results of the FCS assay
clearly demonstrate the inhibitory effect. With a 4-fold excess of
18, the formation of larger oligomers was largely prevented,
even after incubation for 7 days (see Table 2, inhibition).
Results obtained with a 2-fold excess of 18 were still significant.
To rule out other explanations, it is important to look at
particle numbers. The dominance of the fast-moving fraction
might result from precipitation of Aβ by compound 18 to levels
below the aggregation threshold. Alternatively, higher-order
oligomers might have been precipitated selectively so that only
the fast-moving fraction remained in the sample. Both
mechanisms would result in a depletion of dye-labeled Aβ. In
these experiments, however, particle numbers stay constant
over time. As a further control, samples of Aβ (10 μM) and
compound 18 (40 μM) were centrifuged 24 h after incubation.
Particle numbers before and after centrifugation were
unchanged. In contrast, a major drop was seen when this test
was repeated at higher concentrations (100 μM Aβ and 400
μM 18) when precipitation indeed occurs.
a
Table 3. Summary of ThT Measurements
incubation time (days)
control
0
2
7
0 μM 18
0
74
7
100
b
b
b
b
d
inhibition
20 μM 18
40 μM 18
20 μM 18
40 μM 18
nd
nd
nd
nd
15 14
21 15
85 20
80 20
28 11
34
62 10
75
5
c
c
dissolution
7
a
The relative ThT fluorescence with the starting value representing
0% and the maximum after 7 days representing 100% is shown.
Samples were incubated at 37 °C. The mean values of three
b
c
measurements are given. Not determined. Measured after
d
aggregation for 2 days and 1 h after addition of 18. Mean value of
two measurements.
LILBID experiments were conducted at laser desorption
intensities below a level that would lead to dissociation of
noncovalent complexes, to ensure that the mass spectra reflect
the solution distribution of the Aβ oligomers. The concen-
trations employed were 100 μM Aβ and 400 μM 18 (4 °C). As
already mentioned, Aβ and the inhibitor will partially
precipitate under such conditions. The amount of Aβ remaining
in solution, however, is sufficiently high for analysis by LILBID
(>10 μM). Precipitation may also be the cause of difficulties
with the LILBID droplet formation process when incubation
temperatures increased to 37 °C.
For the freshly dissolved sample (Figure 2, blue) as well as
for the sample incubated with compound 18 (red), the spectra
show mainly monomeric Aβ with a small amount of dimer,
trimer, tetramer, and pentamer. In fact, the monomer/oligomer
ratio in the presence of compound 18, calculated according to
In our FCS studies, a solution of Aβ was doped with a small
quantity of its Cy5-labeled analogue. Such samples permit us to
visualize peptide aggregation by confocal microscopy, as well.
When the buffer also contains thioflavin, Aβ fibrils can be
stained by Cy5-Aβ and ThT simultaneously (Figures S7−
S11).51 At time zero, no aggregates could be found in the
CLSM images. After incubation for 2 and 7 days, only a few
aggregates were detected, with both Cy5 and ThT (Figure S7).
The coincidence of red and green fluorescence in the pictures is
important to demonstrate that Cy5-Aβ is integrated into Aβ
fibrils and does not form aggregates by itself. If 100 μM Aβ is
used, at time zero some larger structures can be seen after an
extended search in the droplet. This confirms the findings of
FCS and LILBID (Figure 1C) that even at the beginning of the
aggregation process Aβ samples are not monomeric to 100%.
Influence of Compound 18. To elucidate the activity of
peptidomimetic 18, we examined its effect on the Aβ
aggregation with the different methods. For FCS measure-
I
1
monomer/oligomer =
∑ (Inn)
F
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Figure 4. Time dependence of the monomer/oligomer ratio measured via LILBID. Aβ (100 μM) was incubated for 48 h at 4 °C before the addition
of 400 μM 18. The monomer/oligomer ratios determined 0 min, 70 min, and 48 h after the addition of 18 correspond to the spectra in Figure 3.
The lines serve as only guides for the eye. The inset shows an enlargement of the first 75 min, revealing a lag time before dissolution.
(I is the intensity; n ≥ 2 for the size of the respective oligomer),
increases from an initial value of 1.33 to a final number of 1.87,
which shows an increase in the level of monomeric Aβ in
relation to those of its oligomers. The largest oligomer size
observed decreases from penta- to tetramer. In contrast, the
control sample without the inhibitor (black) shows a
monomer/oligomer value of 0.33 and (Aβ)8 as the largest
oligomer.
The ThT data show a distinct inhibition by 18 as the
fluorescence intensity was reduced to ∼30% (see Table 3,
inhibition). Both concentrations of 18 (20 or 40 μM; 10 μM
Aβ) led to comparable decreases in fluorescence, but the
fluorescence intensity was still higher than at time zero,
indicating that some fibrillar forms must have built up. This is
in accordance with the few structures observed with CLSM (see
below). It can also be seen that the fluorescence intensity after
7 days is higher than after 2 days. We assume that the inhibitor
massively slows Aβ oligomerization as implied by FCS and
LILBID but cannot prevent the slow conversion of medium-
sized oligomers into fibrillar forms. These may have arisen from
minor nucleation sites still present after the preparation of
monomeric Aβ.
The confocal microscopy images showed some small
structures, stained simultaneously by Cy5 and ThT (Figure
S10). As in the Aβ samples incubated without inhibitor (Figure
S7), the number of aggregates is low. A quantitative comparison
by integrating over entire samples is not possible. Instead, we
have analyzed several sectors (67.3 μm × 67.3 μm) where
particles became visible after 7 days using the software package
Fiji.52 The software superimposes the scans measured at 20
different axial positions within the sample droplet (distance
between imaging planes of 0.5 μm) and integrates the
fluorescence of Cy5 and ThT independently. In four different
sectors, ratios of 10.5 1.3 (intensity Cy5/intensity ThT) were
measured in the sample containing 18. In contrast, samples of
Aβ incubated without inhibitor showed a clear shift toward
containing 100 μM Aβ and 400 μM 18 were analyzed by
CLSM, red cloudy structures caused by co-precipitation of Cy5-
Aβ became visible (Figure S11). Such cloudy structures did not
show the green emission of ThT. At lower concentrations (10
μM Aβ and 40 μM 18), precipitation effects could not be
observed (Figure S9).
Dissolution of Preformed Aggregates by Compound
18. Next we employed FCS to investigate the ability of 18 to
dissolve preformed Aβ aggregates. Solutions of Aβ (10 μM)
were kept at 37 °C for 2 days in the absence of the inhibitor.
Then a 2- or 4-fold molar excess of 18 was added. Samples were
examined 1 h after the addition and after an additional 5 days at
37 °C. Within the first hour of incubation with 40 μM inhibitor,
the monomeric fraction of Aβ in the FCS assay increased from
0 to ∼70% (Table 2, dissolution). This effect was less
pronounced for a 2-fold excess, but a significant effect was
still seen. In parallel to the increase in the level of monomers,
particle numbers also increased in these experiments by 1 order
of magnitude. After 5 days in the presence of the inhibitor (7
day experiment), the fast-moving fraction of Aβ amounted to
60% for both concentrations of 18. This demonstrates that 18
can dissolve larger species to monomers and small oligomers.
Nevertheless, the final amount of monomeric Aβ is larger when
compound 18 is present over the whole period of incubation.
In the ThT assay, no significant effect of molecule 18 was
observed as the fluorescence intensity did not change after the
addition of the inhibitor (Table 3, dissolution). Consistently,
CLSM images showed some small aggregates.
LILBID experiments were conducted as before with samples
containing 100 μM Aβ and 400 μM 18 incubated at 4 °C.
Samples were aggregated for 2 days in the absence of the
inhibitor. Then a 4-fold excess of 18 was added, and spectra
were recorded during the first 70 min and 2 days after the
addition of the inhibitor. Already after 70 min, no oligomers
larger than (Aβ)5 could be detected, and after an additional 2
days, the situation was comparable to that in which 18 was
present during the whole aggregation period (Figure 3). The
monomer/oligomer ratio increases from 0.33 before the
ThT emission with ratios of 5.2
3.0 (see page S22).
Accordingly, the fibrillar character of aggregates formed in the
presence of 18 seems to be less pronounced. When samples
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addition of the inhibitor to 0.97 70 min later, which is ∼50% of
the final ratio of 1.62.
Formation of a complex between Aβ and OR1 can be directly
seen in LILBID spectra (Figures S5 and S6), whereas such
signals are missing when Aβ is mixed with 18. This observation
was surprising at first but can be understood in light of the
interaction characteristics, which differ for OR1 and 18. The
lack of signals representing Aβ−18 complexes does not
necessarily indicate weaker binding in the case of 18: Aβ
monomers have a net charge of −3. Thus, in addition to
hydrophobic interactions, ligand binding also has a strong
electrostatic component. Net charges of inhibitors fall from +4
(OR2) and +3 (OR1) to +2 (18). In the LILBID desorption
process, the aqueous droplets undergo an explosive expansion
upon laser irradiation, which sets the solvated ions free. With an
increasing rate of loss of water molecules, ionic attraction more
and more outperforms the hydrophobic effect. Accordingly,
complexes of ligands with high opposite charges have a better
chance to withstand dissociation in the gas phase: Even though
the Arg-Arg-Arg peptide (net charge of +4) does not inhibit the
aggregation of Aβ, the complexes are nicely visible.31 In
The fast dissolution effect of 18 can be seen in Figure 4
where the largest increase in the monomer/oligomer ratio, after
a short lag time, occurs within the first 70 min. For comparison,
the known inhibitor OR115 was also examined, a peptide with
an aqueous solubility higher than that of compound 18
(Figures S5 and S6). Distinct complexes of Aβ oligomers with
one or two molecules of OR1 are visible in the spectra, and Aβ
aggregates are dissolved, as well.
Proteolytic Stability. We tested the resistance against
proteolytic degradation by treatment with proteinase K at 37
°C. Peptide OR1 was completely degraded after incubation for
24 h, whereas 18 showed no sign of instability (Figures S2 and
S3).
Cytotoxicity. Cytotoxicity is an important issue when cell
culture experiments are envisioned. For that reason, the
proliferation of HEK-APPwt cells was examined in the presence
of different concentrations of compound 18.53 In the
concentration range of 30 nM to 10 μM, no toxic effects
were observed. Cell viability, however, dropped to 50% at
concentrations of 30 μM. In addition, we studied the toxicity of
trifluoroacetate and chloride salts of 18 in human neuro-
blastoma cell line SH-SY5Y. Compared to the control, LDH
release was significantly elevated after incubation for 24 h with
18-TFA (20 and 40 μM) and 18-Cl (20 and 40 μM). Metabolic
activity was significantly reduced after incubation for 24 h with
10 μM 18-TFA. Although TFA ions are known for adverse
effects, the impact of 18-Cl on metabolic activity was even
slightly more pronounced (Figure S4).
contrast, the powerful aggregation inhibitor bexarotene,6
a
lipophilic carboxylate with a net charge of −1, does not show
signals for the Aβ complex in LILBID (data not shown).
Given the increased lipophilicity of 18, we cannot strictly
exclude sequestration of Aβ by hydrophobic aggregates of 18.
This mechanism can be responsible for the nonspecific
inhibition of enzymes by certain types of lipophilic com-
pounds.54 Protein aggregation can be blocked in the same
way.55 FCS data, however, provide evidence against hydro-
phobic sequestration: such effects should have increased the
diffusion time and reduced particle numbers.
At concentrations of compound 18 used in our assays, the
viability of HEK-APPwt and SH-SY5Y cells is already
compromised. It is important to note, however, that
concentrations of Aβ used in such cell-free aggregation studies
(10−100 μM) are also far above the physiological levels.
Intracellular aggregation occurs in hydrophobic environments
where both Aβ and inhibitor candidates will be locally
concentrated. In fact, studies using HEK-APPwt cells in vitro
showed beneficial effects of 1 and 10 μM 18. These results will
be reported in due course.
DISCUSSION
■
Starting from the well-known sequence KLVFF, we have
prepared new peptidomimetics that retard the aggregation of
synthetic Aβ1−42. With the first generation of compounds, we
showed that the FF part can be replaced by a condensed
aromatic system and that the terminal amino group is not
necessary for activity. In the second and third generations, the
amount of amino acids in the inhibitor structure was further
reduced. The most simplified candidate 18 resembles the
starting peptide KLVFF only distantly. It has a largely reduced
number of hydrogen bond donors and acceptors. As a
consequence, 18 can hardly form β-sheet-like structures with
Aβ as other peptidomimetics might. 18 bears some similarity to
polyphenolic structures like curcumin and resveratrol, which
also consist of two functionalized aromatic rings connected by a
linker.
Inhibitor 18 is stable against proteolytic degradation by
proteinase K, and the inhibitory activity could be demonstrated
by FCS, ThT fluorescence, LILBID, and CLSM. 18 can largely,
but not completely, prevent Aβ aggregation over extended
periods of time. A slight increase of ThT emission accompanies
the formation of a few particles that can be viewed by CLSM.
Existing aggregates of Aβ can be partially dissolved by both
compound 18 and peptide OR1. However, no significant
decrease in ThT emission is observed. Mature fibrillar
structures thus seem to be more stable against the action of
compound 18. This view is further supported by gel
electrophoresis (Figure S12). The formation of large aggregates
that cannot enter the gel is mostly prevented by 18. Once
formed, however, this material does not disappear when 18 is
added subsequently.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
chem.7b00223.
Full description of synthetic methods, data for character-
izing new compounds, proteolytic stability of OR1 and of
compound 18, determination of solubility and toxicity,
preparation and oligomerization of Aβ peptides, methods
used to monitor Aβ aggregation in the absence and
presence of OR1 and compound 18 (FCS, LILBID, ThT,
1
CLSM, and gel electrophoresis), and H and 13C NMR
spectra of all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: m.goebel@chemie.uni-frankfurt.de.
■
ORCID
Mike Heilemann: 0000-0002-9821-3578
Gunter P. Eckert: 0000-0002-8002-9983
Nina Morgner: 0000-0002-1872-490X
H
DOI: 10.1021/acs.biochem.7b00223
Biochemistry XXXX, XXX, XXX−XXX

Biochemistry
Article
Arrest of beta-amyloid fibril formation by a pentapeptide ligand. J. Biol.
Chem. 271, 8545−8548.
Michael W. Gobel: 0000-0002-5694-4823
̈
Present Address
(14) Re, F., Airoldi, C., Zona, C., Masserini, M., Ferla, B. L.,
Quattrocchi, N., and Nicotra, F. (2010) Beta amyloid aggregation
inhibitors: small molecules as candidate drugs for therapy of
Alzheimer’s disease. Curr. Med. Chem. 17, 2990−3006.
(15) Austen, B. M., Paleologou, K. E., Ali, S. A. E., Qureshi, M. M.,
Allsop, D., and El-Agnaf, O. M. A. (2008) Designing peptide inhibitors
for oligomerization and toxicity of Alzheimer’s beta-amyloid peptide.
Biochemistry 47, 1984−1992.
⊥G.P.E.: Institute of Nutritional Sciences, Justus-Liebig-
University Giessen, Wilhelmstr. 20, D-35392 Giessen,
Germany.
Funding
N.M. is supported by Cluster of Excellence Frankfurt
(Macromolecular Complexes) and received funding from the
European Research Council under the European Union’s
Seventh Framework Programme (FP7/2007-2013)/ERC Grant
agreement n° 337567. T.L. is funded by the DFG-GRK 1986.
(16) Taylor, M., Moore, S., Mayes, J., Parkin, E., Beeg, M., Canovi,
M., Gobbi, M., Mann, D. M. A., and Allsop, D. (2010) Development of
a proteolytically stable retro-inverso peptide inhibitor of beta-amyloid
oligomerization as a potential novel treatment for Alzheimer’s disease.
Biochemistry 49, 3261−3272.
Notes
The authors declare no competing financial interest.
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̈
Tinker, C., Jones, G., Kolosov, O., Salvati, E., Gregori, M., Masserini,
M., and Allsop, D. (2013) A novel retro-inverso peptide inhibitor
reduces amyloid deposition, oxidation and inflammation and
stimulates neurogenesis in the APPswe/PS1ΔE9 mouse model of
Alzheimer’s disease. PLoS One 8, e54769.
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Inhibition of beta-amyloid(40) fibrillogenesis and disassembly of beta-
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ACKNOWLEDGMENTS
■
The authors thank Prof. Martin Grininger (Goethe University
Frankfurt) for providing access to his CCD camera. The
solubility of compound 18-TFA was determined by Frank
Kaiser.
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