BBA - General Subjects 1864 (2020) 129569

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BBA - General Subjects

journal homepage: www.elsevier.com/locate/bbagen

EL ARTICULO INCLUYE ALTERACION DE LA SINTESIS DE LA PROTEINA TAU , SINTETIZADA POR LAS NEURONAS . ESTAS FORMAN EL ARMAZON DE LAS NEURONAS , ESTAS PROTEINAS
TAMBIEN INFLUYEN EN LA MENBRANA CELULAR Y SU FUNCIONAMIENTO. LA RELEVANCIA DEL ARTICULO POR SI APLICACION CLINICA EN ALTERACIONES NEUROLOGICAS .

Modulation of tau protein aggregation using ‘Trojan’ sequences T

⁎
Gaurav Pandey, Sudhir Morla, Sachin Kumar, Vibin Ramakrishnan
Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, India

TAU: PROTEINA
A R T I C LE I N FO A B S T R A C T

Keywords: Background: The abnormal assembly of tau into neurofibrillary tangles has been associated with over 30 de-
Tau bilitating disorders known as tauopathies. Tauopathies affect millions of people worldwide, yet no clinically
Amyloids approved solution for tau aggregation is currently available.
Peptide-based inhibitors Methods: We employed a structure-based design approach to make a series of short peptide-based perturbants
Taupathies
(Trojans), that can interact with the core hydrophobic fragment of tau protein. Through a combination of various
VQIVYK
biophysical methods, serum stability, toxicity, and blood-brain barrier translocation assays, we have assessed the
OBEJETIVO
efficacy of these designed peptides to intervene the aggregation of tau protein fragment.
Results: Our observations suggest that Trojan peptides could modulate the aggregation of the Ac-VQIVYK-NH2
peptide by either accelerating or arresting its self-assembly and reduce the neurotoxicity of the fibrils formed.
TROJANS: MECANISMO EVITAR LA ALTERACION DEL TAU
The designed perturbant peptides showed three essential pre-requisites such as negligible cytotoxicity, high
proteolytic stability in serum, and an ability to cross human blood-brain barrier (BBB). Furthermore, the Trojans
could disassemble the pre-formed fibrillar assemblies.
Conclusions: These designed Trojan peptides can serve as a potential therapeutic option for tauopathies, mod-
ulating post as well as pre-aggregation leading to the diseases condition.
General significance: Tauopathies are a group of over 20 progressive neurodegenerative disorders that affect
millions of people worldwide. The available therapies of tau-linked neurodegenerative syndromes are limited
and mostly symptomatic and therefore there is an urgent need for a cost-effective treatment option. We are
presenting a series of structure-based, de novo designed, short peptides that can potentially modulate tau protein
aggregation.

1. Introduction
Tauopathies is a collective term for a series of progressive neuro-
pathological disorders characterized by the aggregation of abnormally
phosphorylated microtubule-associated protein tau, into paired helical
filaments (PHFs) and neurofibrillary tangles (NFTs) [1]. Therefore, it
invokes significant interest because, tau protein aggregation is one of
the hallmarks of Alzheimer's disease (AD) [2]. Under normal condi-
tions, the primary function of the tau protein is the stabilization of
axonal microtubules, by attaching to its microtubule binding domain.
The hyperphosphorylation of tau in diseased conditions result in the
destabilization of microtubules leading to the inhibition of axonal
transport [3].
Designing specific inhibitors of tau aggregation can aid in deci-
phering the mechanism of tau aggregation mediated cognitive decline
in AD and other tauopathies. The microtubule binding domain of tau
comprises of a proline-rich region followed by repeat domains which
vary from 4, 3 or 2 in number, depending upon the length of the
isoform. These repeat domains interact with microtubules. Two hex-
apeptide motifs; 306VQIVYK311 in the third repeat domain, and
275
VQIINK280 in the second repeat region are known to be critical in
driving the aggregation of the entire tau protein [4,5] (Fig. 1). Recent
studies have shown that modulation of these motifs (e.g., by mutations)
prevents tau from aggregating, under in vitro and in vivo conditions
[6–8]. These hexapeptides also aggregate to form fibrils similar to those
developed by tau and can serve as a model for examining the me-
chanisms that boost tau fibrillation, and also as a screening platform for
the design of tau aggregation inhibitors [9]. Furthermore, inhibitors
that arrest seeding by capping the ends of tau fibrils can prevent the
progression of tau self-assembly. The crystal structure of the steric
zipper form of VQIVYK segment, from the R3 domain, has been utilized
to design structure-based inhibitors of VQIVYK aggregation [10–13].
Further, these experiments showed that VQIVYK aggregation inhibitors
are equally effective in arresting the aggregation of 3-repeat (3R) tau
isoforms under in vitro conditions [13].
Disease-modifying therapies currently available for tauopathies are

⁎
Corresponding author.
E-mail address: vibin@iitg.ernet.in (V. Ramakrishnan).

https://doi.org/10.1016/j.bbagen.2020.129569
Received 2 October 2019; Received in revised form 18 February 2020; Accepted 20 February 2020
Available online 27 February 2020
0304-4165/ © 2020 Elsevier B.V. All rights reserved.
G. Pandey, et al.
Fig. 1. Schematic representation of domain organization of human tau protein.
limited. Recent reports on AD therapy are stressing the crucial role of
tau derived soluble oligomers and neurofibrillary tangles in disease
pathogenesis, which has shifted focus towards the design of tau ag-
gregation inhibitors. Phenothiazine methylene blue (tau protein in-
hibitor) is presently the only drug under phase III clinical trials of this
kind [14]. Failure of molecule-based remedies due to their inherent
toxicity, poor proteolytic stability, high costs, or poor blood-brain
barrier (BBB) permeability, have stimulated a push towards the devel-
opment of non-invasive therapies such as scanning ultrasound [15,16],
electric fields [17–19], transcranial stimulations by magnetism or
electric current [20,21], and IR [22].
Peptide-based inhibitors offering better specificity, high selectivity,
low toxicity, better BBB permeability, high design diversity, and low
accumulation, serve as a useful alternative for natural products.
Currently, 140 peptide-based drugs are in clinical trials, and around
500 have been referred for preclinical assessment [14]. These initiatives
triggered our pursuit towards designing short peptide sequences, tar-
geting the aggregation of core peptide motif of tau protein. Our ex-
perimental results showed that our peptides were capable of reducing
the tau elicit toxicity by accelerating or impeding the self-assembly of
the core peptide-motif of tau.
2. Results and discussion
2.1. Design, synthesis and primary characterization of the peptide inhibitors
Our structure-based design approach was inspired by the ‘Trojan
horse’ strategy employed by Greeks in the battle of Troy, to breach the
otherwise unbreachable defense of Troy. Analogously, we have de-
signed short-peptide based perturbants, which we refer to as the ‘Trojan
peptides’ to penetrate the tight junctions of the model steric zipper
peptide.
As per the rational structure-based design principle, small molecules
targeted to amyloidogenic species must have primary structural moi-
eties for interacting with the core aggregating segment. To achieve the
rational structure-based development, the design scheme of the trojan
peptides closely mimics the sequence of the chosen model aggregating
peptide (A3), a peptide homologous to a sequence within the third
MTBR (microtubule binding region) of tau [8]. The basic idea was to
model short peptides that could non covalently interact with A3 peptide
and prevent it from forming toxic oligomeric or protofibrillar species
either by blocking its aggregation or accelerating it to form non-toxic
mature fibrils. We have made single amino acid substitutions at position
4 (V4T, V4L, V4I). The V4T substitution was directed to modulate the
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BBA - General Subjects 1864 (2020) 129569
Table 1
List of designed, synthesized, and characterized
Trojan peptides (P15-P21) with A3 as the model
peptide. In the table below, ‘Ac’ and ‘NH2’ denote
N-terminal acetylation and C-terminal amidation
respectively while upper case and lower case al-
phabets denote ‘L' and ‘D' amino acids respec-
tively.
Code Peptide sequence
A3 Ac-VQIVYK-NH2
P15 Ac-VQITYK-NH2
P16 Ac-VQILYK-NH2
P17 Ac-VQIIYK-NH2
P18 VQIVYK-NH2
P19 VQILYK-NH2
P20 VQIYK-NH2
P21 vqivyk-NH2
polarity of the sequence, while the focus of V4L and V4I mutations was
to modulate the chain length and topology of the side chain segment.
We also designed the N-terminal truncated analogs to study the
effect of the change in the charge distribution on the aggregation pro-
pensity of the target peptide. Aggregation propensity of non-acetylated
VQIVYK was found to be significantly less as per earlier reports [23].
This prompted us to explore the non-acetylated VQIVYK, V4L, and V4I
mutants, to potentially act a ‘Trojans’ impeding the protofibrils for-
mation. The poly-D chiral vqivyk segment was designed to prohibit the
epitaxial growth of the aggregating segment, by not offering the hy-
drogen bonding functional groups in the desired angle.
To test our hypothesis, we synthesized the model peptide (end-
capped) Ac-VQIVYK-am as well as the designed Trojan peptides. The
peptide sequences and their respective codes are shown in Table 1.
They were synthesized manually by employing the solid-phase peptide
synthesis protocol, using Fmoc-chemistry and characterized for purity
using high-performance liquid chromatography (HPLC) and MALDI-
TOF mass spectrometry (Fig. S1). All the peptides were ≥ 95% pure.
2.2. Assessment of amyloidogenicity
Verification of self-aggregation propensity is the first step in the
protocol for peptide-based therapies. We tested the self-assembly pro-
pensity of all the designed Trojan peptides using Thioflavin T (ThT)
fluorescence, Fourier-Transform Infrared Spectroscopy (FTIR),
Dynamic Light Scattering (DLS), and Field-Emission Transmission
Electron Microscopy (FETEM). The proteolytic stability and cytotoxicity
of the peptides were assessed using serum stability and MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
2.2.1. Aggregation kinetics estimation
Thioflavin T is a dye that attaches to amyloid fibrils and gives an
enhanced fluorescence emission upon binding, and the intensity of
fluorescence is directly proportional to the extent of fibrillogenesis
[24]. The Trojan peptides were dissolved in water and the aggregation
propensity was estimated intermittently up to 16 h, after adding ThT
dye. The results show that all the Trojan peptides (P15-P21) except P17,
have very low fluorescence signal, comparable to blank ThT dye, sug-
gesting the non-amyloidogenic nature of the Trojans. However, P17
peptide showed 17 times higher fluorescence intensity compared to the
model aggregating peptide (A3) at 0th hour (Fig. S2 (a, b)) suggesting
high amyloidogenicity.
2.2.2. Assessment of amyloid morphology
FETEM micrographs of amyloidogenic peptides can provide a qua-
litative impression of fibrillar assemblies. To further verify the self-as-
sembling nature of our designed peptide, we performed FETEM imaging
of the dried peptide samples. No fibrillar assemblies were observed for
G. Pandey, et al.
each of the Trojan peptides except P17, which showed dense fibrillar
structures, a morphology characteristic of amyloidogenic peptides (Fig.
S2 (c)). The FETEM observations complement the ThT assay results and
suggest that all the Trojan peptides except P17 are non amyloidogenic
in nature.
2.2.3. Secondary structure characterization
To probe into the secondary structure conformation of the Trojan
peptides, FTIR spectra of the dried films of the peptide samples were
recorded (Fig. S3). P17 showed a band around 1624 cm−1, character-
istic of β structure which correlates with the intense ThT fluorescence
signal observed. While P16 displayed a signal around 1627 cm−1 and
P19, P20 and P21 showed band around 1635 cm−1, suggesting β
conformation. Peptide P18 has a signal around 1668 cm−1 which is
suggestive of the turns [25]. P15 did not show any characteristic IR
band.
2.2.4. Toxicity estimation study
Minimal cellular toxicity is a pre-requisite for the designed pertur-
bant peptides to be eligible as a potential therapeutic agent against
tauopathies. The cytotoxicity of the Trojan peptides was tested by in
vitro toxicity analysis on Human neuroblastoma SH-SY5Y and IMR-32
neuronal cell lines. The peptide solutions were added to the cells and
incubated for 48 h. Staurosporine (STS) added and untreated cells (CC)
served as positive and negative controls, respectively. The toxicity was
measured using the MTT reduction assay. In case of the SH-SY5Y cells,
the cell viability estimates were more than 84% except for P19 and P21
treated samples, which showed 79% and 72%, respectively. For IMR-32
cells, the viability was more than 88% for all the samples except P17
treated samples, which showed 71% viability (Fig. 4 (a), (b) and Table
S1, S2). Following the initial cytotoxicity measurements, a compre-
hensive cytotoxicity assessment of the designed trojan peptides at
various concentrations was done to estimate their IC50 value (half-
maximum inhibitory concentration). The viability of SH-SY5Y and IMR-
32 were observed to be decreased to less than 50% at trojan peptide
concentrations measuring approximately 500 μM and above (Fig. S4).
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BBA - General Subjects 1864 (2020) 129569
Fig. 2. Effect of the designed Trojan peptides on
model amyloidogenic peptide: Fibrillation kinetics of
A3 examined by ThT fluorescence estimation in the
presence and absence of (a) accelerator peptides, and
(b) inhibitor peptides. Size estimation using dynamic
light scattering (DLS) in the presence and absence of
(c) accelerator peptides, and (d) inhibitor peptides.
2.2.5. Assessment of peptide stability in the presence of serum
The synthetic peptides often lack the conformational stability, a
prerequisite for an adequate drug, therefore, assessment of peptide
stability in serum is an essential screening assay for the elimination of
unstable peptides from the drug development pipeline. The Trojan
peptides were incubated in equal amounts of Fetal Bovine Serum (FBS),
at 37 °C for four hours and Human Serum at 37 °C for twelve hours. Post
incubation, 6% Trichloroacetic acid (TCA) was added to precipitate the
larger serum proteins. The precipitated serum proteins were separated
by centrifugation while the soluble Trojan peptides present in the su-
pernatant were analyzed for their stability using HPLC and MALDI-TOF.
The HPLC chromatograms of serum treated and untreated peptides
showed peaks at identical positions and mass derived from the MALDI
analysis of these HPLC peaks matched with the theoretical mass of the
untreated peptide (Fig. S5 and S6). The additional peaks observed in
the HPLC chromatograms (Fig. S5 and S6) at 28, 38, and 40 min elusion
time, correspond to the serum proteins (Fig. S7). The results of the
serum stability assay indicate that all the designed Trojan peptides are
stable in FBS as well as human serum. The serum stability assay of P17
was not attempted since it showed 17 times higher fluorescence in-
tensity compared to the model aggregating peptide (A3) at 0th hour
(Fig. S2 (b)) suggesting high amyloidogenicity. P17 because of its self-
aggregating nature, may not be an ideal aggregation modulator.
2.3. Effect of designed peptides on model aggregating peptide
We tested the modulatory effect of the designed Trojan peptides on
self-assembly kinetics of Ac-VQIVYK-am (A3). The aggregating peptide,
A3 was allowed to aggregate in the presence and absence of equimolar
concentration of each of the Trojan peptide. The effect of treatment was
monitored using various biophysical, cytotoxicity and blood-brain
barrier (BBB) experiments.
2.3.1. Examining the kinetics of self-assembly using Thioflavin T
fluorescence assay
Peptide self-assembly was monitored using a time-dependent ThT
G. Pandey, et al.
fluorescence assay quantitatively estimating aggregation in treated and
untreated A3 peptide. The fluorescence intensity of untreated A3 pep-
tide increased with time, showing a characteristic sigmoidal curve. The
peptide sample allowed to aggregate in the presence of P15, P16 and
P17 showed 25-fold, 35-fold and 15-fold higher fluorescence intensities
from the 0th hour respectively, suggesting an accelerated aggregation
upon treatment (Fig. 2 (a) and Fig. S8 (a)). On the contrary, treatment
with P18, P19, P20 and P21 Trojan peptides, showed negligible fluor-
escence intensities compared to untreated A3 peptide, indicating an
impeded aggregation (Fig. 2 (b)). Owing to their ability to accelerate or
decelerate the aggregation of the core-peptide segment of tau, A3,
peptides P15, P16 and P17 were designated as ‘accelerators’ while
peptides P18, P19, P20, and P21 as ‘inhibitors'. Interestingly, a con-
centration titration from 0.5× to 4× did not yield any significant
difference in aggregation inhibition (Fig. S9).
2.3.2. Monitoring the size distribution using DLS
The size of amyloid aggregate is known to increase with increased
rate of aggregation [26]. Dynamic light scattering measurements were
performed to compare the size distribution of amyloid-assemblies
formed by peptide sample treated with accelerator and inhibitor pep-
tides compared to the untreated ones. The DLS plot of the untreated A3
peptide sample, indicated the presence of large-sized aggregates (Fig. 2
(c)). In the case of accelerators peptides, the average distribution in-
creased up to one order of magnitude (Fig. 2 (c)) and Fig. S8 (b)).
While, treatment of A3 peptide sample with inhibitor peptides resulted
in ten-fold reduction, in average size distribution (Fig. 2 (d)). The DLS
data complements our observations from ThT fluorescence measure-
ments.
2.3.3. Effect of ‘Trojan peptides’ on the morphology
Field-emission transmission electron microscope (FETEM) imaging
was performed to examine the effects of the designed Trojan peptides
on the morphology of A3 aggregates. The untreated sample (A3) as well
as the sample treated with accelerators (P15, P16 and P17) show the
presence of dense fibrils. Contrastingly, samples treated with inhibitors
(P18, P19, P20 and P21) showed non characteristic structures, and no
fibers were observed, indicating the absence of the amyloids in the
mixture (Fig. 3). The FETEM observations also complement our ob-
servations from ThT assay and DLS measurements and show that ac-
celerator peptides promote the formation of fibrillar self-assemblies
while, inhibitor peptides arrest the self-assembly of A3 into amyloido-
genic fibrils.
2.3.4. Secondary structure elucidation using FTIR
The secondary structure conformations of the untreated and treated
A3 samples was determined by FTIR analysis. While untreated A3
peptide showed a characteristic amide I band of the β-sheet, A3 treated
with peptides P15, P16 and P17 displayed a peak around
1624–1626 cm−1, suggesting that the accelerator treated samples re-
tain β-sheet conformation in dried films (Fig. S10). A3 peptide treated
with Trojans P18, P19, P20, and P21 showed prominent peaks centered
around 1662–1669 cm−1 which are characteristic of non β conforma-
tion, often identified as turns (24). FTIR results suggest, that accelerator
treated A3 samples retained their β confromation while inhibitor
treated samples showed a shift from β to a non β conformation. This
result suggests, that the morphological difference induced by Trojan
treatment is expressed at the supramolecular assembly and at the sec-
ondary structure level, as well.
2.3.5. Effect of ‘Trojan peptides’ on the cyotoxicity
The cytotoxic amyloid assemblies of tau protein aggregates are
considered as the hallmark of tau linked pathogenesis in AD as well as
other tauopathies. The toxic tau aggregates have been ascribed to cause
synaptic dysfunction and neuronal loss [27,28]. Further, an increasing
number of recent reports propose oligomeric intermediates formed
4
BBA - General Subjects 1864 (2020) 129569
during fibrillization, to be more toxic than the mature fibrils [29–31].
Hence, arresting the tau in its non-toxic form or accelerating the self-
assembly to mature fibrillar state may qualify as a therapeutic option.
The impedance or acceleration of A3 self-assembly, confirms the po-
tential efficacy of the designed Trojan peptides for therapeutic appli-
cations in amyloidogenic diseases. Therefore, we measure the en-
hancement in the cell viability as a result of administration of Trojans
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) reduction assay. Human neuroblastoma SH-SY5Y and IMR-
32 neuronal cell lines were treated with 25 μM of Trojan treated and
untreated A3 peptide solution. A3 samples were initially allowed to
aggregate in the presence (treated) or absence (untreated) of an in-
hibitor or accelerator Trojan peptide. The test results show that the
untreated amyloidogenic A3 peptide solution lowered the cell viability
of SH-SY5Y and IMR-32 to 69% and 57%, respectively. The treatment
with Trojan peptides however, resulted in the cell viability improve-
ments from 6% to 30% in SH-SY5Y and 13% to 38% in IMR-32 cell lines
(Fig. 4 (c), (d)) and Table S3, S4). In general, both accelerator and in-
hibitor Trojans positively impact cell viability. However, from our ob-
servations, accelerator Trojan peptides are found to have increased cell
viability compared to the inhibitors. This supports hypothesis that oli-
gomers may be the primary pathogenic entities and the formation of
mature fibrils could be protective step employed by the body [32–36].
2.3.6. Assessment of BBB permeation efficacy of Trojan peptides
One of the prime causes behind the failure of potential antibody-
based and other molecular therapies has been the failure to cross the
blood-brain barrier (BBB) owing to their large size (14). Experimental
studies support the capability of low molecular weight, small hydro-
phobic molecules to cross BBB [37,38]. The small size and hydrophobic
nature of our design scheme can aid the ability of our Trojan peptides in
crossing the BBB. To test this hypothesis, we performed BBB perme-
ability test on an in vitro model of BBB. We used a BBB model com-
prising of human brain endothelial cells, EA.hy926 cultured on the
apical side of a permeable membrane placed between two sections, to
assess the peptide translocation (Fig. S11). Trojan peptide solution was
added on the apical side of the BBB model and allowed to permeate for
5 h. 5(6)-Carboxyfluorescein labelled cell penetrating peptide (CPP),
TAT (47–57) known to have very high unidirectional influx across the
BBB, was used as a positive control [39]. Post incubation, the solution
in the basolateral side was collected. The peptide translocation was
quantified as discussed in materials and methods section. The experi-
mental results show that after 5 h, approximately 24% P15 and P16,
56% P18, 4.4% P20 and 61% P21 translocated across model BBB
system. P19 shows 0% and positive control (TAT (47–57)) showed 83%
translocation across model BBB system (Fig. 4 (e)). The results of BBB
permeability assay confirm the BBB translocation ability of the Trojan
peptides with P18 and P21 showing the BBB permeation comparable to
the TAT (47–57), a well known brain delivery vector.
2.3.7. Estimation of BBB integrity post Trojan treatment
The BBB has an overall protective function, and thus the therapeutic
designs should ensure from any impairment or disruption to the normal
functioning of BBB. Therefore, the integrity of BBB model post-treat-
ment with peptide Trojans was evaluated by estimating FITC fluores-
cence post incubation, to detect any damage to the BBB [40,41]. The
results of BBB integrity evaluation show very low FITC fluorescence for
Trojan peptides, pointing to their negligible influence on the barrier
integrity, and hence no paracellular leakage (Fig. 4 (f)). Positive control
peptide TAT (47–57), however, shows medium to high FITC fluorescence
and indicating a paracellular leakage. Our observation from the BBB
integrity assay show that the Trojan peptides are capable of translo-
cating the BBB, without affecting its interity.
G. Pandey, et al.
Fig. 3. FETEM analysis: FETEM micrographs of Trojan treated and untreated A3 peptide. The untreated A3 sample and the A3 samples allowed to aggregate in the
presence of accelerator peptides (P15, P16, P17) showed fibrillar aggregates. Contrastingly, A3 samples treated with inhibitor peptides (P18-P21) showed non
characteristic morphologies.
2.4. Effect of inhibitor peptide on pre-aggregated A3 peptide
A3 peptide, allowed to aggregate for 12 h and treated with inhibitor
peptides, and ThT fluorescence measurements were recorded at 24th
and 36th hour to investigate the effect of inhibitor peptide on the al-
ready aggregated A3 peptide. We observed that A3 peptide treated with
P18, P19, P20, and P21, showed approximately 10-fold, 8-fold, 10-fold
and 22-fold decrease in fluorescence intensities at 24th hour respec-
tively, compared to untreated A3. After the 36th hour the reduction in
treated A3 fluorescence intensity with P18, P19, P20, and P21, was 13-
fold, 13-fold, 9-fold, and 16-fold respectively (Fig. S12 and Table 2).
The results of this experiments suggest that not only designed peptide-
based inhibitors could prevent the amyloidogenesis of A3 peptide but
they can also disaggregate the pre-formed A3 self-assemblies, and P21
peptide was the most potent amyloid breaker among experimental test
set used in this study.
3. Conclusion
The molecular intervention of amyloidogenesis continues to be the
motivation behind many drug discovery efforts against tauopathies and
several molecular modulators have advanced to animal or human
clinical trials (14). Here, we designed a series of peptides targeting the
minimal amyloidogenic hexapeptide “VQIVYK” (A3). This peptide has
been reported to play a critical role in the abnormal aggregation of tau.
We demonstrated that our peptide-based modulators (Trojans) were
non-amyloidogenic, stable in serum and showed minimal cytotoxicity
on two model neuronal cell lines SH-SY5Y and IMR-32. The treatment
of A3 with acetylated Trojan peptides accelerated the rate of its self-
assembly, while the non-acetylated counter-parts prevented A3 from
self-assembling. Soluble oligomers and protofibrils have been widely
accepted to be the key toxic species in the case of amyloidogenic pep-
tides. Through the results of the present study, we hypothesize that
aggregation elicited cytotoxicity can be overcome by either arresting
the self-assembly of amyloidogenic peptides in their monomeric forms
or by shifting the equilibrium to stable fibrillar aggregates, away from
5
BBA - General Subjects 1864 (2020) 129569
toxic oligomeric species in solution by accelerating the rate-limiting
nucleation step. These observations are in agreement with several re-
cent reports, wherein an accelerated aggregation has been demon-
strated to diminish the toxicity of amyloid self-assemblies [42–48]. The
effect the designed peptides on the size of aggregates, morphology,
secondary structure conformation and cytotoxicity of the A3 peptide
was also investigated. The treatment of A3 with Trojans impeded the
formation of large aggregates and prevented the formation of toxic fi-
brillar morphologies, an observation also supported by FETEM experi-
ments. FTIR experiments show that the Trojans can modulate secondary
structure conformation of the target peptide and therefore suggest their
effect is not just limited to the assembly level. The results of MTT assay-
based cytotoxicity studies showed that there are up to 38% improve-
ment in the cell viability of the peptide-treated A3 samples, while
preliminary BBB permeability experiments demonstrated the BBB
crossing ability of the peptides. The addition of Trojan peptides to pre-
aggregated A3 samples, lowered the extent of aggregation. The eight
experiments discussed in this paper validates the efficacy of Trojan
peptides in modulating the aggregation of tau protein, reducing the
aggregation induced cytotoxicity. Stability in serum, BBB permeability
and ability to break pre-formed self-assemblies, further add value to the
designed ‘Trojan peptides’ to be used as a future therapeutic option.
4. Experimental procedures
4.1. Materials
All the chemical reagents were purchased from industrial suppliers
and used without any further purification. Fmoc-protected amino acids
and N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexa-
fluorophosphate (HBTU), 1-Hydroxybenzotriazole hydrate (HOBt) were
purchased from Novabiochem (Darmstadt, Germany). Rink amide resin,
N,N-diisopropylethylamine (DIPEA), piperidine, trifluoroacetic acid
(TFA), thioanisole, ethanedithiol, acetic anhydride, and thioflavin T
(ThT), were bought from Sigma-Aldrich. N,N-dimethylformamide,
diethyl ether, and m-cresol were obtained from Merck. 1,1,1,3,3,3-
G. Pandey, et al. BBA - General Subjects 1864 (2020) 129569

Fig. 4. (a, b) Trojan toxicity estimation: The inherent toxicity of designed peptide Trojans was estimated by adding them to SH-SY5Y, and IMR-32 cells and
determining the cytotoxicity was determined using an MTT assay. (c, d) Effect of peptide Trojans on the cellular toxicity of A3 peptide: Post incubation, A3 samples
were allowed to aggregate in the presence and absence of Trojan peptides, and were subsequently added to SH-SY5Y, and IMR-32. The cytotoxicity effect was
determined using MTT assay. The molecular perturbation reduced the toxicity of A3 peptide evident from the increased cell viability compared to the untreated
sample. Additional data for the cell viability is available in Table S3 and S4 of supporting information. (e) BBB translocation assay: An in vitro BBB model comprising
of an of immortalized human brain endothelial cell line EA.hy926, grown as a monolayer on the apical side of the Transwell plate. The percentage translocation was
estimated by measuring the Tyr absorbance and was directly proportional to it. The measurements were compared to two controls: one an empty Transwell (“Filter”)
and Tat (47–57) peptide (positive control). Peptides P21 and P18 showed the best BBB permeability. (f) BBB integrity assay: Any loss of integrity of BBB model due to
the effect of Trojan peptides was evaluated by adding a high molecular, FITC labelled FD40 compound and recording the FITC fluorescence to detect any damage to
the membrane permeability. The Trojan peptides showed minimal fluorescence indicating negligible effect on the barrier integrity. The p-value was calculated with
respect to staurosporine (a, b), and A3 (c, d) using a two-sample, Student's t-test (assuming unequal variances). The p-value of less than 0.05 was considered
statistically significant (*, P < .05, **, P < .05, and ***, P < .001).

Table 2 peptide was further characterized by MALDI-TOF mass spectrometry

Summary of ThT assay data showing inhibition of pre-formed A3 self- (Bruker autoflex speed) using HCCA (alpha-Cyano-4-hydroxycinnamic
assemblies upon treatment with the designed Trojan peptides. acid) matrix.
Experimental condition % Aggregation inhibition
4.3. Preparation of peptide solution
A3 + P18_24h 89.85
A3 + P19_24h 88.02
A3 + P20_24h 89.54 The peptides were treated with 1,1,1,3,3,3-hexafluoro-2-propanol
A3 + P21_24h 95.55 (HFIP) prior to making stock solutions in deionized water to prevent
A3 + P18_36h 92.42 any pre-aggregation. The stock peptide solutions were prepared in
A3 + P19_36h 92.91
deionized water by weighing the dried form of the peptide and dissol-
A3 + P20_36h 88.97
A3 + P21_36h 93.74 ving at a concentration of 1 mM.

4.4. Thioflavin T fluorescence assay

hexafluoro-2-propanol (HFIP), hydrochloride was procured from TCI
Chemicals. The cell lines were procured from the National Centre for
Cell Science (Pune, India). All other reagents were of the highest grade
available. Deionized water was used.
4.2. Peptide synthesis and characterization
Solid-phase peptide synthesis using Rink amide resin
(Novabiochem) and standard Fmoc chemistry was employed to syn-
thesize the peptides manually. The peptides were cleaved from the resin
and deprotected using a cleavage mixture comprising of trifluoroacetic
acid/m-cresol/thioanisole/ethanedithiol in 20:2:2:1 ratio, for 12 h at
ambient conditions. Ice-cold diethyl ether was used to precipitate the
cleaved peptide. The purity of peptides was verified on Shimadzu
Prominence HPLC system using C−18 analytical column. The purity of
ThT binding assay was performed by combining 7.5 μL, 1 mM
peptide to a solution of 10 μM ThT in 50 mM sodium phosphate buffer
pH 7.4. ThT fluorescence was recorded at 482 nm (emission slit
width = 5 nm) for 60 s at regular time intervals on a spectro-
fluorometer (Jasco FP 8500) at 25 °C using a 1 cm path length quartz
cuvette (Helma, Sigma-Aldrich). The excitation wavelength was set at
450 nm (slit width at 2.5 nm).
4.5. Dynamic light scattering (DLS)
To determine the size distribution of aggregates formed in the
presence and absence designed Trojan peptides, the size estimation
assay was performed using dynamic light scattering method. The size
distribution was measured on a Zetasizer Nano ZS (Malvern, UK)

6
G. Pandey, et al.
instrument, equipped with a 633 nm laser and 173° non-invasive
backscattering detector configuration. Size distribution were recorded
for 16 h incubated peptide samples at 25 °C. The representative in-
tensity-weighted hydrodynamic diameters of the samples were plotted
based on 10 scans.
4.6. Field-emission transmission electron microscopy (FETEM)
An aliquot of 10 μL peptide sample was deposited on a carbon
coated 200-mesh copper grid and negatively stained by addition of 2%
uranyl acetate aqueous solution for 1 min. Excess solution was blotted
out from the periphery using a Whatman I filter paper after 2 min. The
sample were air dried. FETEM images were recorded using an Ultra
High-Resolution TEM microscope (JEOL, Model: 2100F).
4.7. Fourier transform infrared spectroscopy (FTIR)
FTIR spectra were recorded on a Shimadzu IRAffinity-1S Fourier
Transform Infrared Spectrophotometer equipped with a diamond ATR
crystal. Aliquots of 10 μL were spread out and dried as films on the
crystal surface and allowed to dry under ambient conditions. All spectra
were recorded in the frequency region 1400–1800 cm−1, with a re-
solution of 4 cm−1 and 64 scans per sample.
4.8. Cell viability assay
All the stock peptide solutions were prepared in deionized water by
weighing the dried form of the peptide and dissolving at a concentra-
tion of 1 mM. The aggregating peptide, A3, was allowed to aggregate in
the presence and absence of equimolar concentration of each of the
trojan peptides for a duration of 16 h. Post incubation, the aqueous
solutions of treated and untreated A3 samples were added to human
neuroblastoma SH-SY5Y and IMR-32 neuronal cell lines and MTT as-
says were performed to assess the cytotoxicity of treated and untreated
Ac-VQIVYK-am. The SH-SY5Y and IMR-32 cells were maintained in a
T25 culture flask containing Dulbecco's modified Eagle's medium
(DMEM). All the cells were supplemented with 10% Fetal Bovine Serum
(FBS), 100 U/mL penicillin and 100 U/mL streptomycin at 37 °C under
5% CO2 and were maintained for further assay by subsequent re-cul-
turing. Prior to the MTT assays, each of the cells (80 μL) were cultured
in 96-well plates at a density of 5 × 103 cells/well for 24 h in a CO2 cell
culture box (Forma SteriCycle CO2 Incubator 371, Thermo Scientific,
USA). Then, the cells were treated with 20 μL control as well as treated
peptide samples (25 μM) and for 48 h. Post incubation, 10 μL MTT
solutions (5.5 mg/mL) were added into each well. The medium was
replaced with 100 μL DMSO to dissolve the formazan crystals after 4 h.
To estimate the cell viability under respective treatments, absorbance at
570 nm was measured by a multifunctional microplate reader (TECAN
Austria GmbH, Grödig, Austria). Six replicates were performed, and the
data were averaged.
4.9. Blood brain barrier transwell permeability assay (in vitro)
An in vitro BBB model comprising of a monolayer immortalized
human brain endothelial cells (EA.hy926) growing on the apical side of
a permeable membrane placed between two compartments was used to
evaluate peptide translocation. Human brain endothelial cells
(EA.hy926) (ATCC, CRL-2299) were cultured in DMEM supplemented
with 10% FBS and 100 U/mL penicillin and 100 U/mL streptomycin in
a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cells were
adhered in monolayers and, after reaching confluence were harvested
using trypsin-EDTA and 3500 cells/well were then seeded in Transwell
filter insert plates (1 μm pore size) (Corning). Cells were allowed to
reach a confluent monolayer (5–6 days) until full confluency was
reached and media was changed every two days. This resulted in the
establishment of stable tight layer mimicking the natural BBB (Fig.
7
BBA - General Subjects 1864 (2020) 129569
S11). Trojan peptide solution was added on the apical side of the BBB
model and allowed to permeate for a duration of 5 h. 5(6)-
Carboxyfluorescein labelled cell penetrating peptide (CPP), TAT (47–57)
known to have very high unidirectional influx across the BBB, was used
as a positive control for BBB permeability in our experimental model.
Post incubation the solution in the basolateral side was collected. The
peptide translocation was quantified by measuring the absorption for
tyrosine present in each of the Trojan peptides and employing the Beer-
Lambert law and the following relation (ii):
Cf
ii⎞⎟ %translocation = x 100
⎠ Ci
where, Cf is concentration of peptide recovered from the basolateral
side of BBB model, post incubation of 5 h, Ci is the initial concentration
of peptide sample added to the apical side of BBB. For estimating the
translocation of 5(6)-Carboxyfluorescein labelled TAT (47–57), fluour-
escence intensity was measured and the percentage translocation was
calculated using following equation:
Ff
(iii)%translocation = x 100
Fi
where, Ff is fluorescence intensity of TAT (47–57) recovered from the
basolateral side of BBB model, post incubation of 5 h, Fi is the initial
concentration of the same solution added to the apical side of BBB.
4.10. Blood brain barrier integrity evaluation assay
The integrity of BBB model post treatment with peptide Trojans was
evaluated by adding fluorescein isothiocyanate (FITC)-conjugated
dextran (FD40, with molecular weight of 40 kDa) as a probe to Trojan
pre-treated wells. A 25 mg/ml stock of FD40 was prepared in water and
was later diluted in DMEM without phenol red, to final absorbance
below 0.1. Diluted FD40 probe was added to the apical side for 2 h. Post
incubation FITC fluorescence was estimated at 493 nm and emission
ranging from 500 nm to 600 nm using a spectrofluorometer (Jasco FP
8500), to detect any damage to the BBB.
4.11. Serum stability assessment
The stock peptide solutions were prepared in deionized water by
weighing the dried form of the peptide and dissolving at a concentra-
tion of 1 mM. For assessing their serum stability, the peptides were
incubated with an equal volume of Fetal bovine serum and human
serum for four and twelve hours respectively. Post incubation 6% TCA
(trichloroacetic acid) was added to the peptide-serum mix to precipitate
the serum proteins, followed by incubation at 4 °C for 15 min. After
15 min, the TCA treated the peptide-serum mixture was centrifuged at
13000 rpm for 10 min. The supernatant was analyzed using a Shimadzu
Prominence HPLC system equipped with a C-18 analytical column to
determine any serum-mediated proteolysis in the sample [49]. The
stability of peptide was further characterized by MALDI-TOF mass
spectrometry (Bruker autoflex speed) using HCCA (alpha-Cyano-4-hy-
droxycinnamic acid) matrix.
4.12. Pre-formed aggregate study
To investigate the effect of inhibitor peptide on the pre-formed A3
aggregates, A3 peptide allowed to aggregate for 12 h. It was then
treated with inhibitor peptides, and ThT fluorescence measurements
was recorded, as described above at 24th and 36th hour.
Author contribution
VR designed the experiment. GP synthesized peptides and per-
formed the biophysical experiments. GP and SM performed the cell
G. Pandey, et al.
culture and BBB experiments. VR, GP analyzed, interpreted data, and
contributed to the editing of the manuscript. SK, SM and GP analyzed
and interpreted cell viability assay data. GP and VR analyzed the results
and wrote the paper in close collaboration with all the authors. VR
conceived & directed the ideas, planning and overall execution.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgment
We acknowledge Board of Research in Nuclear Sciences,
Department of Atomic Energy, Govt. of India (35/14/07/2017-BRNS)
and Department of Biotechnology, Govt. of India (BT/565/NE/U-Excel/
2016) for funding. We thank Central Instruments Facility, IIT Guwahati
for instrument support.
Note: Patent on peptide-based modulators of aggregation, No.
TEMP/E-1/36478/2019-KOL.
Appendix A. Supplementary data
MALDI plots of synthesized peptides (Section 1), ThT assay profiles
and TEM micrographs of Trojan peptides (Section 2), FTIR spectra of
Trojan peptides (Section 3), Cell viability assay data of Trojan peptides
(Section 4), Serum stability analysis data (Section 5), ThT assay profiles
and DLS plot of P17 treated A3 peptide (Section 6), Aggregation ki-
netics profiles of Trojan treated A3 peptide (Section 7), FTIR spectra of
Trojan treated A3 peptide (Section 8), Cell viability assay data of Trojan
treated A3 peptides (Section 9), Schematic of blood-brain barrier model
(Section 10), ThT assay profiles of pre-aggregated A3 peptide treated
with Trojans (Section 11), and Statistical Appendix (Section 12).
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bbagen.2020.129569.
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