Peptidotriazolamers Inhibit Aβ(1–42) Oligomerization and Cross a Blood-Brain-Barrier Model

Article abstract of DOI:10.1002/cplu.202000814

In peptidotriazolamers every second peptide bond is replaced by a 1H-1,2,3-triazole. Such foldamers are expected to bridge the gap in molecular weight between small-molecule drugs and protein-based drugs. Amyloid β (Aβ) aggregates play an important role in Alzheimer's disease. We studied the impact of amide bond replacements by 1,4-disubstituted 1H-1,2,3-triazoles on the inhibitory activity of the aggregation “hot spots” K¹⁶LVFF²⁰ and G³⁹VVIA⁴² in Aβ(1–42). We found that peptidotriazolamers act as modulators of the Aβ(1–42) oligomerization. Some peptidotriazolamers are able to interfere with the formation of toxic early Aβ oligomers, depending on the position of the triazoles, which is also supported by computational studies. Preliminary in vitro results demonstrate that a highly active peptidotriazolamer is also able to cross the blood-brain-barrier.

Full text of DOI:10.1002/cplu.202000814

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Peptidotriazolamers Inhibit Aβ(1–42) Oligomerization and
Cross a Blood-Brain-Barrier Model
Nicolo Tonali+,*[a, b] Loreen Hericks+,[a] David C. Schröder,[a] Oliver Kracker,[a]
Radosław Krzemieniecki,[a] Julia Kaffy,[b] Vadim Le Joncour,[c] Pirjo Laakkonen,[c]
Antoine Marion,[d] Sandrine Ongeri,[b] Veronica I. Dodero,[a] and Norbert Sewald*[a]
In peptidotriazolamers every second peptide bond is replaced
by a 1H-1,2,3-triazole. Such foldamers are expected to bridge
the gap in molecular weight between small-molecule drugs and
protein-based drugs. Amyloid β (Aβ) aggregates play an
important role in Alzheimer’s disease. We studied the impact of
amide bond replacements by 1,4-disubstituted 1H-1,2,3-tria-
zoles on the inhibitory activity of the aggregation “hot spots”
K16LVFF20 and G39VVIA42 in Aβ(1–42). We found that peptido-
triazolamers act as modulators of the Aβ(1–42) oligomerization.
Some peptidotriazolamers are able to interfere with the
formation of toxic early Aβ oligomers, depending on the
position of the triazoles, which is also supported by computa-
tional studies. Preliminary in vitro results demonstrate that a
highly active peptidotriazolamer is also able to cross the blood-
brain-barrier.
Introduction
genesis of AD is under debate. Besides the most widely
accepted amyloid cascade[4] and cross-interaction[5,6] hypothe-
ses, other assumptions were suggested: tauopathies, cholinergic
neuron damage, oxidative stress, inflammation, microbiota
influencing the immune and endocrine system, and genetic
disposition.[2,7] According to the amyloid cascade hypothesis,
amyloid β (Aβ) aggregates play an important role in the
pathological progression of AD. Aβ spontaneously self-asso-
ciates into soluble oligomers and insoluble aggregates like
protofibrils and fibrils with high β-sheet content.[4] In the past
20 years, the Aβ plaques were considered the main target for
therapeutic development and inhibitors of Aβ aggregation
were designed to reduce the formation of insoluble fibers.[8]
However, all therapeutics reaching phase III clinical trials failed
so far, which led to questioning the role of Aβ and amyloid
deposition in AD pathology.[9–11] Increasing evidence showed
that the small and soluble Aβ oligomers rather than the
insoluble aggregates are particularly cytotoxic and contribute
to either neuronal death and/or affect synaptic neurotrans-
mission.[12] This consideration allowed to reconsider the amyloid
cascade hypothesis and underlines the early aggregation of Aβ
to act as a critical trigger in the etiopathogenesis of the disease.
Aβ oligomers are responsible for the disruption of the learning
behavior in rats and that can also trigger events such as
oxidative damage, inflammation, and calcium deregulation.
Moreover, Aβ oligomers have been shown to also induce tau
hyperphosphorylation, leading to neurite degeneration.[13,14] In
this context, shifting treatment to a pre-symptomatic stage
appears as an appealing and relevant alternative. Therapeutic
strategies intervening at the early oligomerization process
rather than at the later fibrillation step have indeed attracted
attention recently.[15] Among the various strategies which have
been adopted to stop or revert the progression of the disease,
the modulation or inhibition of the aggregation process of Aβ
has been approached through different mechanisms: stabiliza-
tion of its native state,[16] bypassing the on-pathway oligomer
Degenerative disorders due to misfolding and assembly into
various aggregate structures involve actually more than 30
human proteins leading to at least 20 serious human diseases,
named amyloidoses.[1] Among them, Alzheimer’s disease (AD)
can be included as a calamitous neurodegenerative amyloidosis
inducing progressive cognitive decline, functional impairment,
and loss of independence.[2] The number of people worldwide
suffering from AD is expected to reach 75 million by 2030, while
no treatment is available to stop or even slow down the
progression of the disease.[3] The approved drugs only provide
symptomatic relief and short-term benefits. To date, the patho-
[a] Dr. N. Tonali,+ L. Hericks,+ D. C. Schröder, O. Kracker, R. Krzemieniecki,
Dr. V. I. Dodero, Prof. Dr. N. Sewald
Organic and Bioorganic Chemistry
Department of Chemistry Bielefeld University
PO Box 100131, 33501 Bielefeld (Germany)
E-mail: norbert.sewald@uni-bielefeld.de
nicolo.tonali@universite-paris-saclay.fr
[b] Dr. N. Tonali,+ Dr. J. Kaffy, Prof. Dr. S. Ongeri
BioCIS
CNRS, Université Paris Saclay
5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry (France)
E-mail: nicolo.tonali@universite-paris-saclay.fr
[c] Dr. V. Le Joncour, Prof. Dr. P. Laakkonen
Research Programs Unit, Translational Cancer Medicine Research Program
University of Helsinki
00014 Helsinki (Finland)
[d] Assist. Prof. Dr. A. Marion
Department of Chemistry
Middle East Technical University
06800 Ankara (Turkey)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cplu.202000814
© 2021 The Authors. ChemPlusChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
Non-Commercial NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-
commercial and no modifications or adaptations are made.
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formation, inhibition of the fibril elongation and disaggregation
of the already formed amyloid aggregates.[17–20] Several natural
polyphenols have been reported to exhibit potent inhibitory
action on Aβ aggregation,[21] but none of them was able to
demonstrate improvement in cognitive faculties and efficacy on
disease regression during clinical trials.[22] Generally, the main
challenge in inhibiting protein-protein interactions is the
modulation of large and relatively flexible surface areas.[23] The
lack of affinity and selectivity of small molecules, when
employed to modulate this type of interactions, is often related
to this problem.
Aducanumab is a promising monoclonal antibody that is
currently under consideration for approval by the FDA and
EMA. It holds potential for becoming the first treatment for
reducing clinical decline in AD owing to its ability to target
both forms of Aβ, i.e., soluble oligomers and insoluble
fibrils.[24,25] A major drawback of monoclonal antibodies is,
however, the need for repeated administrations and the
associated cost of production.[26]
Research in the protein-protein interaction field has been
also oriented towards peptides, because they offer several
advantages such as greater efficacy, specificity and selectivity,
owing to their intermediate size between small molecule drugs
and protein therapeutics.[27,28] Moreover, the risk of complication
caused by their metabolites is reduced, thus making them safer.
Recently, we reported a new BODIPY-based real-time assay
suitable for 96-well plate format that allows screening of
compounds as selective inhibitors of the formation of Aβ(1–42)
oligomers.[17] We were able to discover new active peptide
inhibitors of the early oligomerization of Aβ(1–42). These
peptides were derived from the two Aβ(1–42) aggregation “hot
spots” K16LVFF20 and G38VVIA42, which form the hydrophobic
central core and the C-terminal domain, respectively. The all-d-
configured acetylated analog Ac-klvff-NH2 1 (Figure 1) proved
to be able to interfere with both oligomerization and fibrilliza-
tion, while the C-terminal fragment H-GVVIA-NH2 2 (Figure 1)
proved to be a specific inhibitor of the oligomerization. Both 1
and 2, therefore, show an ability to protect cells from cytotoxic
dary structures frequently involved in protein-protein interac-
tions by maintaining at the same time the specific side chains
of a peptide sequence.[31–33] In this regard, peptidomimetic
foldamers represent a pharmacologically important class of
compounds, as they are inspired by the structural features of
their bioactive peptide counterparts. Some of us have already
demonstrated that peptidomimetic foldamers are able to inhibit
more efficiently the amyloid aggregation and to preserve the
non-toxic monomer species, thanks to their preorganization as
β-hairpins[18,19,34] or α-helices.[35]
Inspired by the triazolamers pioneered by the Arora
workgroup,[36] which showed encouraging results as protease
inhibitors, the synthesis of a new class of peptidomimetics
containing 1,4- or 1,5-disubstituted 1H-1,2,3-triazoles and amide
bonds in an alternating fashion was reported by us.[37,38] These
peptidomimetics, which were named peptidotriazolamers, can
be considered as hybrid foldamers with features of peptides
and triazolamers, with conservation of the amino acid side
chain. In 2014, Johansson et al. described the synthesis of
several achiral peptidotriazolamers of various lengths, compris-
ing 1,5-disubstituted triazoles and peptide bonds in an
alternating pattern.[39] Their conformational analysis and quan-
tum chemical calculations showed the concurrence of various
conformers with comparable stabilities.[39] Based on new
synthetic approaches toward enantiomerically pure propargyl-
amines with stereogenic centers in the propargylic position, we
reported on synthesis and conformational analysis of homo- or
heterochiral peptidotriazolamers.[37,38] In the case of 1,5-disub-
stituted peptidotriazolamers the homochiral analog forms a
compact β-turn-like structure, while the heterochiral one with
alternating chirality adopts a polyproline I-like conformation.[37]
Investigation of conformational properties of 1,4-disubstituted
peptidotriazolamers by molecular dynamics simulations, using
specifically tailored force field parameters, suggested a well-
defined and significantly different secondary structure for the
homo- (helix) and heterochiral (twisted S) variants in DMSO.[38]
Aβ(1–42) oligomers. Despite their effectiveness, the l-config- Results and Discussion
ured peptides are generally sensitive to proteolytic degradation
and lack a specific secondary conformation, which might
increase their affinity and specificity for Aβ(1–42).
In the present study, we investigated the potential application
of 1,4-disubstituted peptidotriazolamers for the design of new
inhibitors of Aβ(1–42) oligomerization. In particular, the impact
of the amide bond substitution by 1,4-disubstituted 1H-1,2,3-
triazoles on the inhibitory activity of the two “hot spots”
K16LVFF20 and G39VVIA42 was of interest. These sequences
correspond to the previously described peptides 1 and 2
(Figure 1).[17]
We substituted two amide bonds in both sequences in an
alternating fashion and thus obtained five new peptidomimet-
ics 3–7 (Figure 2): 3, 4, and 6 with the substitution at the
second and fourth amide bonds, while 5 and 7 at the first and
third ones. The two K16LVFF20 peptidotriazolamers 3 and 4, with
l- and d-configuration respectively, were designed in order to
evaluate the possible influence of the stereochemistry on the
inhibitory activity. These new compounds were assessed in the
complementary ThT and BODIPY fluorescence assays and in a
Over the past decade, foldamers have increasingly attracted
attention as useful tools to mimic secondary structures of
proteins and peptides. Bio-inspired synthetic foldamers provide
effective biopolymer mimics with new and improved physio-
logical properties, and represent useful tools for practical
applications in the areas of diagnostics or therapeutics.[29,30]
Peptidomimetic foldamers offer the possibility to adopt secon-
Figure 1. Parent peptides Ac-klvff-NH2 1 and H-GVVIA-NH2 2.[17]
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Scheme 1. Synthesis of propargylamines 12a–d using Ellman’s auxiliary.
Figure 2. Chemical structure of the five peptidotriazolamers 3–7 discussed in
this study.
cell viability assay in order to evaluate their ability to maintain
or even improve the inhibitor activity on the fibrillization and/or
oligomerization of Aβ(1–42), as their peptide counterparts. This
structure-activity relationship study together with molecular
dynamics simulations allowed us to identify the best position
for the triazole rings in the peptide sequence and the
conformational secondary structure requirement to modulate
the protein-protein interactions. Finally, we investigated the
ability of the best peptidotriazolamer to cross the blood-brain-
barrier (BBB) to assess its potential use in vivo.
Scheme 2. Synthesis of propargylamines 16a–d according to the Bestmann-
Ohira approach.
In the Bestmann-Ohira approach, the carboxylic acid of the
(S)- or (R)-Phe and Lys was first reduced to the alcohol 14 (80-
90% of yield for Phe and 40% for Lys) (Scheme 2), which
subsequently was oxidized in a Swern oxidation to give the
corresponding aldehydes 15, which were used without further
purification for the next step. The latter was then converted
into the propargylamines (16b–d) using the Bestmann-Ohira
reagent (yields over two steps of 80% for Phe and 53% for Lys).
For propargylamine 16a, the aldehyde was obtained by
reduction of the Weinreb amide 13 (Scheme 2) by LiAlH4. The
acid labile Boc or Trityl groups were successively cleaved and
replaced by Fmoc to obtain the final propargylamines (16a–d),
suitable for SPPS.
The synthesis of chiral α-azidoesters (18a–g, Scheme 3)
preferably makes use of the pool of naturally occurring amino
acids that can be converted employing Wong’s procedure,
using CuII-catalysed diazo transfer from triflyl azide.[43] The
azides were synthesized in excellent yields of >87%, starting
from the corresponding commercially available amino acid
benzyl ester salts.
Triazole formation was achieved by CuAAC in the presence
of CuSO4 ·5H2O (1.2 eq) and sodium ascorbate (2.4 eq) in
tBuOH/H2O mixture to afford ten 1,4-disubstituted 1H-1,2,3-
triazoles (compounds 19a–j) as dipeptide mimetic building
blocks with satisfactory yields (40-90%) (Scheme 3). Afterwards
the benzyl ester was hydrogenolyzed with triethylsilane in
methanol to obtain the corresponding intermediates (20a–j)
suitable for SPPS (Scheme 3). The peptidomimetics 3–7 were
assembled according to the building block strategy in SPPS by
Synthesis
Peptidotriazolamers 3–7 were synthesized by employing a
building block strategy where the triazole is formed from the
propargylamine and the azide by copper-catalyzed azide-alkyne
cycloaddition (CuAAC) and then coupled by amide bond
formation in solid phase peptide synthesis (SPPS).[40]
Triazoles substituted with adjacent stereogenic centres
require enantiomerically pure propargylamines and α-azido
acids.[41] The (S)À or (R)À configured propargylamines, bearing
the Val (12a,b) and Leu (12c,d) side chains, were obtained
using Ellman’s auxiliary as described by Wünsch et al.[42]
(Scheme 1), while the (S)À or (R)À configured propargylamines
16a–d bearing the Ile, Phe and Lys side chains, are accessible
by Bestmann-Ohira reaction (Scheme 2). In the Ellman’s auxiliary
approach, condensation of the corresponding aldehydes 8 with
(S)- or (R)-configured tert-butyl sulfinamide led to the enantio-
merically pure sulfinylimines 9, which were reacted with
(trimethylsilyl)ethynyllithium, to give the intermediates 10. The
terminal TMS group was successively cleaved to provide the
Bus- (tert-butyl sulfinyl) protected propargylamines 11, with
satisfactory yields (50-60%) and a diastereomeric ratio of 97:3.
The Bus group was then replaced by Fmoc as required for SPPS
(Scheme 1).
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Scheme 3. Synthesis of the dipeptide peptidotriazolamers (20a–j) according to the Bestmann-Ohira approach.
employing Rink amide resin as solid support in order to install a
carboxamide at the C-terminus.
Fluorescence spectroscopy assays
A general synthetic scheme of the peptidotriazolamer is
given in Scheme 4. The dipeptide building blocks or the amino
acid were loaded on the resin by using DIC/Oxyma and collidine
in a mixture of DCM and DMF. After Fmoc removal, the
dipeptide intermediate was coupled under the same conditions.
For the peptidotriazolamers having the natural amino acid at
the N-terminus (d- or l-Lys for 3 and 4 and Gly for 6), the final
amino acid was also coupled by using DIC/Oxyma and collidine.
Peptidomimetics 3, 4, and 5 were acetylated at the N-
terminus before cleavage from resin, while for foldamers 6 and
7 the free N-terminal amine was afforded by directly removing
the Boc N-protecting group under the acidic cleavage protocol.
All peptidotriazolamers, 3–5 as KLVFF mimetics and 6 and 7 as
GVVIA mimetics, were obtained in moderate to good yields
after purification by reverse-phase HPLC.
Three calculated parameters from the BODIPY- and the ThT-
fluorescence kinetic curves can be used to compare the activity
of the inhibitors: t1/2, which is defined as the time at which the
fluorescence has reached 50% of its maximum (as a measure of
the process rate), the slope of the elongation phase of the curve
in the first 4 h of the BODIPY kinetics (as a measure of the
process rate), and F, which is the fluorescence value of the final
plateau and is assumed to depend on the number of
aggregates and fibrils formed, respectively for the BODIPY and
the ThT experiment.[17] Each parameter is defined as the
experimental result in the presence of the tested compound
relative to the one obtained without the compound and is
evaluated as percentage. The presence of oligomers under the
measurement conditions of the BODIPY fluorescence assay has
been already demonstrated by various biophysical assays in our
previous work.[17]
In the ThT assay, the five peptidotriazolamers 3–7 displayed
modest to no inhibitory activity on the fibrillization process
(Figure 3, A, C and E, and Figures S16–20 in the Supporting
Information). In particular, in the KLVFF series the t1/2 values as a
measure of the fibrillization rate in the presence of 3, 4, and 5
(À 47%<t1/2 <14%) were lower than t1/2 of the parent peptide
1, which significantly delayed fibril formation (t1/2= +154%,
Table 1 and S1). Although the parent peptide 2 in the GVVIA
series was inactive on fibril formation, replacing the second and
the fourth amide bound by a triazole ring resulted in the potent
inhibitor 6 of the fibrillization process. The rate was increased
by 50% and the plateau decreased by 52% for 6 at compound/
Aβ(1–42) ratio of 10/1 (Table S1 and Figure 3 C). This effect was
maintained on the fluorescence plateau at a 6/Aβ(1–42) ratio of
1/1 (ΔF=À 39%). However, this inhibitory effect was not
observed for analogue 7 (Figure 3, E), bearing the triazole rings
in the first and third amide bonds position, suggesting that
these positions are critical for inhibition of fibril formation.
The BODIPY fluorescence assay revealed that all designed
peptidotriazolamers (3–7) interfere with early oligomer forma-
tion (Table 1 and S1). Concerning the peptidotriazolamers
based on the KLVFF sequence, the substitution of two amide
bonds by a triazole ring in the second and fourth positions (3
Scheme 4. General synthetic scheme for the SPPS of the peptidotriazolamers
3, 4 and 6 (A) and for 5 and 7 (B), employing the building block strategy.
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Figure 3. Time course of ThT and BODIPY fluorescence showing Aβ(1–42) (10 μM) fibrilliization (A), (C), (E) and oligomerization (B), (D), (F) in the absence (grey
curves) and in the presence of compounds 5 (A) and (B), 6 (C) and (D) and 7 (E) and (F) at compound/Aβ(1–42) ratios of 10/1 (purple curves) and 1/1 (red
curves) and 0.1/1 (blue curve, only for compound 7 in the oligomerization assay). The curves are provided as the average curves of a triplicate.
Table 1. Effects of compounds 3–7 at compound/Aβ(1–42) ratio of 1:1 on 10 μM Aβ(1–42) fibril formation and oligomerization, as assessed by ThT- and
BODIPY-fluorescence spectroscopy, respectively.[a]
ThT assay
F
BODIPY assay
F
Compound
t1/2
Slope
[compound/Aβ ratio]
extension/reduction
[%]
extension/reduction
[%]
reduction
[%]
reduction
[%]
1
2
3
4
5
6
7
n.e.
n.e.
+154�23
n.e.
À 84�0.4
À 80�0.3
À 43�1.8
À 20�0.6
À 81�1.2
À 15�2.9
À 88�1.0
À 94�0.4
À 9�0.8
À 19�2.7
+27�11
+32�17.8
À 39�6.6
n.e.
À 47�9.8
À 36�12.6
À 14�6.7
À 9�0.4
À 24�5.7
À 37�0.6
À 16�1.3
À 88�0.4
À 14�0.4
À 93�0.4
[a] All the compounds were compared with the values obtained for Aβ(1–42) alone (t1/2, F, and slope) and the ones for compounds 1 and 2, already published
in Tonali et al.[17] Parameters are calculated from the expressed as mean curves, as derived by statistical analysis of data after triplicate measurements for each
condition and at least two independent experiments. n.e.: no effect (For further details about calculation, see the Supporting Information).
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and 4) resulted in a reduced inhibitory activity at equimolar
ratio (compound/Aβ(1–42) 1:1) compared to the parent
peptide 1. Compounds 3, 4 and 5 showed very good and
similar efficacy at the highest ratio compound:Aβ(1–42) of 10:1
(Figure S21-S23). Compound 3, with all-(S)-configuration and
triazoles replacing the second and fourth amide bond positions,
showed to be more effective in reducing the oligomerization
rate at a compound/Aβ(1–42) ratio of 1:1 than the all-(R)-
configured analog 4 (À 37% for 3, À 16% for 4, Table 1) as well
as the amount of soluble oligomers (À 43% for 3, À 20% for 4)
(Table 1, Figure S21–22). The positioning of the triazole rings as
substitutes of the second and fourth amide bonds seems to
require an inversion of the stereochemistry, compared to the
peptide 1, to maintain the activity. However, keeping the (R)-
configuration and “shifting” the triazole position to the first and
third amide bonds as in compound 5 resulted in an increase of
the inhibitory effect (À 88% of oligomerization rate reduction
and À 81% of fluorescence decrease at a ratio 1:1, Table 1),
suggesting a possible role of the triazole ring on the conforma-
tional behavior of the peptidotriazolamer (Figure 3, B). The
same observation could be made for the foldamers based on
the Aβ(1–42) C-terminal sequence (GVVIA): compound 7 (Fig-
ure 3, F) is, in fact, more active compared to 6 (Figure 3, D) and
is able to inhibit the oligomerization process of Aβ(1–42) at
both 10:1 and 1:1 ratio (À 93% of oligomerization rate
reduction and À 88% of fluorescence decrease at ratio 1:1,
Table 1), like its peptide counterpart 2. Interestingly, compound
7 maintained a good inhibitory activity even at substoichiomet-
ric ratio (0.1:1) (Figure 3, F), suggesting an important role of the
peptidotriazolamer in stabilizing the correct and necessary
conformation for the interaction with the amyloid peptide,
particularly when triazoles are in place of the first and the third
peptide bonds in a pentapeptide sequence.
Figure 4. Cell viability assay results, representing the percentage of survival
observed for cells incubated without Aβ(1–42), with only inhibitors (25 μM),
and with 5 μm Aβ(1–42) with or without the different inhibitors at different
ratio. A statistically significant difference to Aβ(1–42) treated cells is
indicated by *p<0.05, **p<0.01, and ***p<0.001; n=6 for each condition.
Aβ(1–42), are superior in inhibiting the formation of toxic early
oligomers.
No concentration dependence of effects between 1:1 and
0.1:1 ratios for compound 7 has been found, thus suggesting
that the slight perturbation of the oligomerization process at
0.1:1 ratio observed in the BODIPY assay (Fig. 3F, blue curve) is
enough to produce a protective effect against Aβ(1–42) toxicity
similar to the 1:1 ratio. Further studies will be necessary to
explain the mechanism of action.
Conformational analysis
The conformational preferences of compounds 2, 6, and 7 were
investigated by means of molecular dynamics (MD) simulations
in explicit solvent, to shed light on the differences in activity
observed experimentally and on the related impact of the
triazole positioning in the GVVIA sequence. Following the
strategy designed for our previous studies on peptido-
triazolamers,[37,38] we used a tailored set of molecular mechanics
parameters[44] and obtained the starting structures of MD
simulations by performing a series of gas phase simulated
annealing (SA) conformational samplings with NMR-derived
NOESY interproton distance restraints. Restraints were then
released, and simulations were performed in DMSO to address
the validity of our models with respect to NMR results and in
water to emulate a more biologically relevant environment. In
both solvents, the N-terminal amine was modelled as proto-
nated, with a trifluoracetate (TFA) or a chloride counterion for
DMSO or water simulations, respectively, since the compound
was obtained as a TFA salt and analyzed as such in NMR
experiments. All 200 ns MD simulations were repeated three
times to increase convergence of the results. Additionally, the
same methodology was applied to a set of starting structures
obtained from SA sampling without any interproton distance
restraints. Both approaches yielded similar results, suggesting
convergence of the conformational sampling and all results are
reported in the Supporting Information. In the following, we
Cell viability assays
The most active compounds in the oligomerization assay, 5 and
7, were tested in an MTS viability assay to confirm the effective
rescue of SH-SY5Y neuroblastoma cells in the presence of toxic
Aβ(1–42) oligomers. Their activities were compared to the one
of a previously published N-acetylated peptide (Ac-GVVIA-NH2)
which is active only at a 5:1 ratio, but its protective effect is lost
at equimolar ratio (1:1), thus behaving as positive and negative
control at the same time.[17] Compounds 5 and 7 did not show
any toxicity when incubated alone with cells at 25 μM
concentration (Figure 4). The addition of compound 7 showed a
significant protective effect on the cells from cytotoxic Aβ(1–42)
oligomers at both equimolar and substoichiometric ratios, while
compound 5 was significantly effective at both 5:1 and 1:1
ratios (5/Aβ(1–42)) (Figure 4). These results demonstrate that
the replacement of the first and third amide bonds in the “hot
spot” sequences of Aβ(1–42) is an efficient bioisosterism
strategy to obtain new peptidomimetics with comparable
activity. Moreover, the substoichiometric inhibitory activity of 7
compared to 5 proved that peptidotriazolamers, designed for
the C-terminal sequence instead of the hydrophobic core of
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shall only focus on results obtained from MD run with NMR-
based SA starting structures.
peptide 2 populates two to three conformations on the
Ramachandran plots, the introduction of triazole rings peptide
bond isostere significantly rigidifies the backbone of com-
pounds 6 and 7. Consistently with the analysis of RMSD and
RoG time series, the nature of the solvent has little to no effect
on the conformational distribution of the three compounds.
Structural clustering based on backbone RMSD was per-
formed to extract representative conformations of the three
compounds (Figure 5) and to examine the impact of the amide
bond isostere on the backbone conformation. The main clusters
of all compounds were found to be highly stable with
populations ranging from 48 to 99% in DMSO and from 74 to
81% in water. In the upper panel of Figure 5, the individual
overlay of DMSO and water representative structures for each
compound confirms the low impact of the solvent on the
preferred conformation. Compounds 2 and 7 adopt a similar,
extended conformation, while peptidotriazolamer 6 folds in a
“U”-shape. Interproton distances derived from NMR spectra
were compared with those measured over all frames of the
major cluster in DMSO. For all three molecules, average
deviations considering all interproton distances were found to
be as small as 0.8 Å, which validates the predicted models
Analysis of the root mean square deviation (RMSD) and
radius of gyration (RoG) time series (Figure S26) suggest that
the simulations reached equilibrium and that the conforma-
tional sampling converged for all molecules. In general, the
backbone of each compound is mainly responsible for the
fluctuations of RMSD in both solvents. The natural peptide 2
and to some extent the peptidotriazolamer 7 have a more
flexible backbone compared to compound 6 in both solvents.
For each compound, there is no significant difference in the
RMSD and RoG time series when moving from one solvent to
another, expect for compound 2, which seems to adopt a
slightly more extended conformation in water when comparing
its DMSO and water RoG time series.
Distributions of Φ/Ψ torsion angles pairs are depicted in
the Supporting Information (Figure S27). Although a direct
comparison of dihedral angles is not applicable when dealing
with natural and modified peptides due to the differences in
torsion angle definitions, a qualitative analysis of the conforma-
tions on a single amino acid basis reflects significant differences
between the compounds. While each amino acid in the native
Figure 5. Upper panel: representative structures for the major backbone cluster of compounds 2, 6 and 7 in DMSO (orange) and Water (iceblue); lower panel:
superimposition of the representative structure of compounds 2 and 6 as well as 2 and 7 in water.
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against experimental observations. The major deviations were
associated with distance pairs involving hydrogen atoms in
sidechains. A detailed analysis is available in Table S2. The
similarities between 2 and 7 extend to the spatial orientation of
their hydrophobic side chains, which overlap well except for
the N-terminal part, resulting a similar signature for intermo-
lecular interactions. On the contrary, compound 6 presents a
significantly different spatial organization of its polar and non-
polar chemical functions.
Solute-water radial distribution functions (RDFs) are de-
picted in Figure 6 for four selected atomic centers on the solute
backbone. In all molecules, the glycine amino proton interacts
with one water molecule yielding a characteristic peak for a
hydrogen bond centered at about 1.8 Å. The triazole nitrogens
N2 and N3 can serve as hydrogen bond acceptors and the
corresponding distributions indeed peak at about 2.0 Å. How-
ever, as already pointed out in our previous studies on 1,4-
peptidotriazoles,[38] the peak of the first solvation shell is broad
and very low in intensity, indicating that the solvent is loosely
interacting with these peptide bond surrogates. The RDF of the
amine proton of alanine shows a sharp peak at 1.8 Å.
This peak integrates for one water molecules in the case of
compound 2 and 7, and for a substantially smaller fraction for
compound 6, indicating that the N-terminal part of the latter is
somewhat less solvated than that of the two other molecules.
Overall, even for the strongest interactions discussed for the
three molecules, the RDF plots are characteristic of compounds
with a significant hydrophobic character. This is in line with
Lipinski’s rule of five and its requirement for a certain degree of
lipophilicity, which may be beneficial to promote the BBB
passage of the present drug candidates.
oligomerization.[45] This structure, named cluster03b by the
authors, was shown to form penta- and hexamers that remain
stable over long MD simulation times. Figure 7B presents the
main conformation of compounds 2, 6, and 7 obtained in water
aligned to the N-terminal part of Aβ(1–42) according to Tran
et al. As illustrated in the panel A of Figure 7, G38VVIA42 is
involved in a β-sheet conformation that allows an exact match
of hydrophobic side chains with the rest of the sequence and
that contributes to the overall stability of the structure. Align-
ment of 2, 6, and 7 to G38VVIA42 shows that 2 and 7 can
recapitulate both the conformation and the lipophilic signature
of the C-terminal fragment of Aβ(1–42). Compound 6, however,
yields a very poor alignment due to its bent conformation.
This last observation is in line with the experimental BODIPY
assay (Table 1) and highlights the similarities between com-
pounds 2 and 7. It also suggests a rationale for the significantly
lower activity of compound 6 obtained in the assay. The
intrinsically extended conformation of compounds 2 and 7
allows them to readily substitute G38VVIA42 in the oligomer
forms of Aβ(1–42), while 6 adopts a conformation in solution
that is incompatible. Compound 6 would need to undergo a
significant conformational change in order to bind in place of
G38VVIA42. Considering the high stability of the most populated
structures for all three compounds in water, it can be inferred
that such conformational change would be energetically
demanding, which would prevent binding of 6 or significantly
slow it down compared to 2 and 7.
Tran et al. had identified, via extensive replica exchange
molecular dynamics, a structure of Aβ(1–42) that promotes
Figure 7. Possible mode of action of compounds 2 and 7 as aggregation
inhibitor. A) Ring shaped 42 residue beta amyloid monomer (cluster03b
from Tran et al.).[45] Beside the overall structures depicted in cartoon style,
relevant side chains and backbone atoms stabilizing the β-sheet are
highlighted in liquorice style. B) RMSD based structural alignment of
compounds 2 (top), 6 (middle) and 7 (bottom), depicted in liquorice style, to
the GVVIA-sequence, shown in cartoon style. The hydrophobic groups are
depicted in CPK style.
Figure 6. Radial distribution function for selected solute-water interactions.
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In vitro blood-brain-barrier permeability measurements
a promising model for future design of drugs targeting the
central nervous system.
Encouraged by the solute-water RDF results, we investigated
the in vitro permeability of peptidotriazolamer 7, the most
active compound of the series, in order to evaluate its ability to Conclusion
cross an in vitro artificial blood-brain-barrier (aBBB). As quanti-
fied by HPLC-MS, 7 was found to cross the artificial BBB,[46]
consisting of murine brain microcapillary or immortalized
human umbilical vein endothelial cells co-cultured with
astrocytes from the same species (Figure 8 and S28). These
models mimic the physical properties of the actual BBB in
addition to showing low permeability values even for passively
diffusing molecules (Figure S28B). According to a previous
similar assay,[47] 8 h for the collection of samples showed to be a
relevant timepoint. Kinetics studies are possible in this BBB
model assay, but require to repeatedly take the cells out of the
incubator. The change of temperature and atmosphere is
detrimental to the stability of the barriers inducing a very
significant increase in the barrier permeability. This would
compromise determining the actual ability of the studied
compound to cross the barrier. We decided to use the
peptidomimetic in 1 mM concentration to warrant the detec-
tion after crossing the BBB model. Lower doses might have
caused problems with the detection limit even when using
highly sensitive methods like LC-MS-MS, leading to biased
conclusions. The presented data were obtained from triplicates
of two independent biological models using either human or
mouse cells to establish the barriers. The outcome was
comparable in these two models, with a low variability in the
values among the triplicates, thus proving the significance of
the results.
We designed and synthesized peptidotriazolamers containing
1,4-disubstituted 1H-1,2,3-triazoles based on the two hot spot
sequences K16LVFF20 and G39VVIA42 of Aβ(1–42) as parent
peptides. The building block strategy of dipeptide mimetics by
SPPS synthesis proved to be a versatile and adaptive method to
prepare peptidomimetic foldamers characterized by synthetic
monomers mimicking natural dipeptide side chains. By per-
forming fluorescence assays and cell viability assays, we proved
their ability in reducing the Aβ(1–42) oligomerization and the
amount of soluble toxic oligomers formed. Peptidotriazolamer
7 was found to be an inhibitor with similar activity as its
peptide counterpart and retained good inhibitory activity even
at a substoichiometric ratio (7/Aβ(1–42)=0.1:1). On the other
hand, compound 6, which differs from 7 only in the position of
the triazole moieties, did not show any inhibition of oligomeri-
zation.
Conformational analyses by molecular dynamics simulations
allowed to shed light on the differences in activity observed
experimentally for compounds 2, 6, and 7 and on the related
impact of the triazole positioning in the sequence. We
demonstrated that peptidotriazolamer 7 can adopt an extended
conformation like the parent peptide 2, while peptidotriazo-
lamer 6 folds in a “U”-shape. This different conformational
behavior can be ascribed to the triazole positioning, which
affects the global secondary structure of the pentapeptide
sequence and thus its ability to disrupt the protein-protein
interactions during the Aβ(1–42) oligomerization process.
In conclusion, we demonstrated the practical application of
peptidomimetic foldamers in the area of therapeutics, partic-
ularly in pathologies involving abnormal protein-protein inter-
actions. Peptidotriazolamers represent a new important class of
peptidomimetic foldamers with biological activity. The further
demonstration that this type of foldamers is also able to cross
the in vitro BBB provides supporting data that peptidotriazo-
lamers hold promise for the future design of drugs targeting
the central nervous system.
As shown in Figure 8, compound 7 is able to cross both
murine and human BBB models, with a greater extent observed
for the latter one. 8 h after addition of 1 mM dosage, 7�0.3%
of compound 7 had crossed the murine barrier, while 29�1.7%
were detected for the human barrier. This result demonstrated
that peptidotriazolamers are able to cross the artificial human
BBB to a higher extent than the murine one, thus making them
Experimental Section
Synthesis of peptidotriazolamers
For the synthesis of the building blocks 20a–j and all their
intermediates, see the supporting information.
Resin was first swollen in DMF for 1 h in a reaction vessel equipped
with a sintered glass bottom. Fmoc group on the linker was then
removed by using a solution of 0.1 M HOBt and 20% piperidine in
DMF (8 mL) for 40 min (deprotection cycle was performed twice).
Subsequently, Fmoc protected amino acid or dipeptide triazolamer
(building blocks 20a–j) was first activated in situ by treatment with
DIC (1.1 eq.) and Oxyma Plus (1.1 eq.) in DCM for 10 min and then
transferred to the resin, dispersed in a solution of DMF (4 mL) and
Figure 8. Passage of peptidotriazolamer 7 through the in vitro mouse (grey)
and human (green) BBB at 8 h after addition of 1 mM dosage on the upper
chamber. Data shown are means from three different triplicates�SE.
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2,4,6-trimethylpyridine (10 eq.). The amino acid or the building
block (20a–j) was then coupled by mechanical shaking at ambient
temperature overnight. The coupling step was followed by a
capping phase, using a solution of acetic anhydride (10 eq.) in DMF
(8 mL) and stirring for one hour at ambient temperature. Successful
completion of each coupling was monitored by UV spectroscopic
method. The cycle of Fmoc removal and coupling was repeated
with subsequent Fmoc protected amino acids or building blocks
20a–j to obtain resin-bound desired peptidotriazolamers (3–7).
Each coupling was followed by removal of Fmoc, washing the resin
by dimethylformamide (5×8 mL), dichloromethane (3×8 mL),
methanol (1×8 mL), dichloromethane (1×8 mL) and dietylether
(1×8 mL) and then drying. Cleavage of peptides from resin was
carried out by using TFA:triisopropylsilane:water (95:2.5:2.5) as
cleavage cocktail (10 mL), stirred by mechanical shaking at ambient
temperature for 2 hours. Filtration afforded the peptidomimetic in
filtrate which was evaporated under vacuum to remove the excess
of TFA. Then, the crude was dissolved in ACN/water 1:1 (v/v) and
freeze-dried, before performing the purification by HPLC (0%À 70%
of ACN/H2O/TFA 95:5:0.1 in 45 min).
performing a double orbital shaking of 10 s before the first cycle.
Experiments were started by adding Aβ(1–42) to final concentration
of 10 μM into a mixture containing 0.53 μM BODIPY in 20 mM
buffer (pH 7.4) with and without the tested compounds at different
°
concentrations (100 and 10 μM) at 25 C. The kinetic curves are the
representative average of triplicate measurements and representa-
tive values of two independent experiments. The ability of
compounds to inhibit Aβ(1–42) aggregation is reported in Table 1
considering the time of the half-life of aggregation (t1/2), the
intensity of the experimental fluorescence plateau (F) and the slope
of the linear part of the curve (first 4 hours). Refer to the Supporting
Information for further details concerning the calculation of all the
parameters.
Cell viability assay
SH-SY5Y neuroblastoma cells were grown in low serum Optimem
°
(Life Technologies) for 24 h at 37 C, 5% CO2 in a 96 well plate at 20
000 cells per well. Aβ(1–42) was prepared according to the
oligomerization protocol B (see the Supporting Information). The
Aβ(1–42) aliquot was prepared by dissolving in sterile PBS (20 mM
phosphate buffer pH 7.4) reaching a final Aβ(1–42) concentration of
50 μM in the presence of 250 and 50 μM of compound 5 and of 50
Fluorescence-detected Thioflavin-T binding assay
°
Stock solutions of compounds (3–7) were prepared in DMSO
(20 mM). The Aβ(1–42) peptide was prepared in an aqueous 1%
ammonia solution to a concentration of 1 mM and then, just prior
to use, diluted to 0.2 mM with 10 mM Tris-HCl, 100 mM NaCl buffer
(pH 7.4). Thioflavin T fluorescence was optimized to evaluate the
development of Aβ(1–42) fibrils over time using a fluorescence
plate reader (Fluostar Optima, BMG lab-tech) with standard 96-well
black microtiter plates. The ThT fluorescence intensity was recorded
with 440/480 nm excitation/emission filters set for 42 h applying a
double orbital shaking of 10 s before the first cycle. Experiments
were started by adding Aβ(1–42) reaching a final concentration of
10 μM into a mixture containing 40 μM Thioflavin T in 10 mM Tris-
HCl, 100 mM NaCl buffer (pH 7.4) with and without the tested
and 5 μM of compound 7 for 24 h at 37 C, along with a negative
control without inhibitor and a positive control with Ac-GVVIA-NH2
(250 and 50 μM). After the 24 h period, media was removed from
the cells and replaced with Optimem containing the preincubated
Aβ(1–42) solutions diluted one to ten (5 μM Aβ(1–42) final
concentration). The cells were incubated for a further 24 hours. The
cell viability (MTS assay) was performed using the Cell Titer 96®
Aqueous One Solution Cell Proliferation Assay (Promega). The assay
was performed in two independent experiments with n=6 for each
condition. The significant difference between the values from cells
in the presence of Aβ(1–42) and cells in the presence of Aβ(1–42)
and the inhibitor (p<0.05, 0.01, and 0.001) was measured by
statistical ANOVA test.
°
compounds at different concentrations (100, 10 and 1 μM) at 25 C
(maximal DMSO concentration of 0.5% (v/v)). Each condition was
recorded in triplicate and the kinetic curve was fitted to a Boltzman
sigmoidal equation using GraphPad Prism 7 from which the time of
Conformational analysis
Molecular Dynamics (MD) simulations were conducted using the
Amber20 modelling software suite and the specially tailored TZLff
molecular mechanics force-field parametrization for peptido-
triazolamers.[44,48] Initial 100 independent simulated annealing MD
runs in gas phase with NMR derived distance restraints served as
input for the subsequent MD simulation in explicit DMSO environ-
ment as well as TIP4Pew water. After energy minimization and
heat-up of the solvated system, three parallel 200 ns MD produc-
tion runs for each molecule and solvent were performed at 300 K.
All non-time dependent analyses were performed on merged
replica data over the last half of each trajectory. Time series were
analyzed for each replicate individually for the whole trajectory
length. Further details regarding the methodology can be found in
the Supporting Information.
the half-life of aggregation (t1/2) and the intensity of the
experimental fluorescence plateau (F) were obtained. The ability of
compounds to inhibit Aβ(1–42) aggregation was assessed consider-
ing both the t1/2 extension/reduction and the F reduction. Refer to
the Supporting Information for further details concerning the
calculation of all the parameters.
Fluorescence-detected BODIPY binding assay
Stock solutions of the BODIPY dye for spectroscopic measurements
and for time-dependent kinetics were prepared in EtOH
(0.0428 mM) and subsequently diluted to 5.3 μM in PBS buffer
(stock solution). The Aβ(1–42) peptide was dissolved during 15–20
minutes at room temperature in 0.16% NH4OH solution at a
peptide concentration of 2 mg/mL ans successively dried by
immediate lyophilization. Then, the Aβ(1–42) peptide was recon-
In vitro blood-brain barrier permeability measurements
°
stituted in 1% NH4OH (at 1 mg/mL) with sonication at 25 C for 1
In vitro mouse and human BBB inserts were prepared as described
by Le Joncour et al.[46] Briefly, human BBB is induced by co-culturing
immortalized HuAR2T endothelial cells[49] with normal human
astrocytes (Lonza, CC-2565) for 7 days. Mouse BBB is obtained by
co-culturing the brain microvessel endothelial cell line (bEND3)[50]
with HIFko mouse astrocytes for 5 days.[51] As described in Le
Joncour et al.[46] and in Fig. S28 A (SI), assay quality verification is
performed before testing the molecule of interest. Briefly, once the
optimal co-culture incubation duration for establishing the barriers
minute before diluting in 20 mM phosphate buffer pH 7.4. Stock
solutions of compounds (3–7) were prepared in DMSO (20 mM) and
later diluted in PBS buffer to reach two different concentration
(400 μM and 40 μM). BODIPY fluorescence was optimized to
evaluate the development of Aβ(1–42) oligomers over time using a
fluorescence plate reader (Tecan i-control 200) with standard 96-
well black microtiter plates. The BODIPY fluorescence intensity was
recorded with 518/540 nm excitation/emission filters set for 9 h
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Acknowledgements
We acknowledge Carmela Michalek for the cell viability assay. The
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The authors declare no conflict of interest.
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Keywords: αβ oligomerization inhibitors
foldamers · peptidomimetics · peptidotriazolamers
·
amyloids
·
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FULL PAPERS
Oligomerization inhibition: Peptido-
triazolamers are foldamers compris-
ing 1,4-disubstituted 1H-1,2,3-
Dr. N. Tonali*, L. Hericks, D. C.
Schröder, O. Kracker, R. Krzemieniecki,
Dr. J. Kaffy, Dr. V. Le Joncour,
Prof. Dr. P. Laakkonen, Assis-
t. Prof. Dr. A. Marion, Prof. Dr. S.
Ongeri, Dr. V. I. Dodero, Prof. Dr. N.
Sewald*
triazoles as substitutes of each
second peptide bond. Sequences
designed on the basis of amyloid β
(Aβ) are able to modulate the Aβ(1–
42) oligomerization process and to
cross a blood-brain-barrier model.
1 – 13
Peptidotriazolamers Inhibit Aβ(1–
42) Oligomerization and Cross a
Blood-Brain-Barrier Model