Sunil Bansal1, Indresh Kumar Maurya2, Nitin Yadav3, Chaitanya Kumar Thota3, Vinod Kumar1, Kulbhushan Tikoo1, Virander Singh Chauhan3, Rahul Jain1. 1. Department of Medicinal Chemistry and Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S Nagar, 160 062 Punjab, India. 2. Department of Microbial Biotechnology, Punjab University, Sector 14, Chandigarh 160 014, India. 3. International Center for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Abstract
Since the introduction of acetyl cholinesterase inhibitors as the first approved drugs by the US Food and Drug Administration for Alzheimer's disease (AD) in clinics, less than satisfactory success in the design of anti-AD agents has impelled the scientists to also focus toward inhibition of Aβ aggregation. Considering the specific binding of fragments for their parent peptide, herein, we synthesized more than 40 new peptides based on a C-terminus tetrapeptide fragment of Aβ1-42. Initial screening by MTT cell viability assay and supportive results by ThT fluorescence assay led us to identify a tetrapeptide showing complete inhibition for Aβ1-42 aggregation. Peptide 20 displayed 100% cell viability at 20 μM concentration, while at lower concentrations of 10 and 2 μM 76.6 and 70% of cells were viable. Peptide 20 was found to restrict the conformational transition of Aβ1-42 peptide toward β-sheet structure. Inhibitory activity of tetrapeptide 20 was further evidenced by the absence of Aβ1-42 aggregates in electron microscopy. Peptide 20 and other significantly active tetrapeptide analogues could prove imperative in the future design of anti-AD agents.
Since the introduction of acetyl cholinesterase inhibitors as the first approved drugs by the US Food and Drug Administration for Alzheimer's disease (AD) in clinics, less than satisfactory success in the design of anti-AD agents has impelled the scientists to also focus toward inhibition of Aβ aggregation. Considering the specific binding of fragments for their parent peptide, herein, we synthesized more than 40 new peptides based on a C-terminus tetrapeptide fragment of Aβ1-42. Initial screening by MTT cell viability assay and supportive results by ThT fluorescence assay led us to identify a tetrapeptide showing complete inhibition for Aβ1-42 aggregation. Peptide 20 displayed 100% cell viability at 20 μM concentration, while at lower concentrations of 10 and 2 μM 76.6 and 70% of cells were viable. Peptide 20 was found to restrict the conformational transition of Aβ1-42 peptide toward β-sheet structure. Inhibitory activity of tetrapeptide 20 was further evidenced by the absence of Aβ1-42 aggregates in electron microscopy. Peptide 20 and other significantly active tetrapeptide analogues could prove imperative in the future design of anti-AD agents.
First reported by Alois
Alzheimer in 1906, Alzheimer’s disease
(AD) is a form of dementia that currently affects nearly 46.8 million
people worldwide, and about 10% of those are in United States alone.[1] Such a huge number of patients create numerous
challenges for the society.[2] Because the
acetyl cholinesterase inhibitor drugs tacrine, donepezil, rivastigmine
and galantamine, and N-methyl-d-aspartate-receptor
antagonist memantine were approved by the Food and Drug Administration
(FDA) in 1990’s with the last being in 2003, not significant
progress has been witnessed in the last decade. Though some secretase
inhibitors, immunotherapeutics, metal chelators, and tau aggregation
inhibitors have been through to some phases of clinical testing, most
of them could not reach advanced stages and very few progressed to
phase III trials and later failed owing to a number of reasons.[3−9] Amongst a plethora of factors reported playing roles in the etiology
of the disease, accumulation of amyloid β (Aβ) plaques
is the most widely accepted phenomenon.[10−12] Though inhibition of
Aβ aggregation currently remains one of the viable target for
the global scientific community,[13,14] the designing
of anti-amyloidogenic agents is no exception in being associated with
own variety of challenges.[15] A number of
small molecules have shown potential to disrupt the Aβ aggregation
process and few even progressed to clinical trials; however, their
nonspecific mechanism of action have restricted their further developments.[16−18] Bexarotene, a small molecule and an USFDA approved drug for cancer,
is recently reported to block initial nucleation steps of Aβ
assembly and delay the onset of toxic aggregates has completed phase
II clinical trials.[19]Because peptide
fragments are specific in their binding with their
parent peptide, the very initial focus on the design of Aβ inhibitors
revolved around the sequence derived peptide fragments of Aβ
itself. Pioneer work in this regard mentions the inhibitory effects
of a pentapeptide KLVFF (Aβ16–20) fragment
derived from the central hydrophobic sequence of full-length peptide.
Since then, the following one and a half decade focused on the design
of inhibitors based on the modification of this pentapeptide sequence.
As recently reviewed by Han and He,[15] a
number of modifications such as introduction of d-amino acids
and other unnatural residues, incorporation of hydrophilic oligomers
at ends, retro-inversion, C-terminal amidation, and use of β-sheet
interrupting residues were reported to exhibit anti-Aβ aggregation
properties.[20−25] Modified peptides such asSEN304 have undergone preclinical studies
and PPI-1019 and iAβ5 were tested in phase II clinical trials;
however, the studies were halted.[26,27] An N-methylated
pentapeptide, SEN-606, is currently in clinical trials.[28] Gozes and co-workers reported an octapeptide,
NAPVSIPQ (NAP), derived from activity-dependent neuroprotective protein
that interfered with Aβ self-assembly and disaggregated preformed
fibrils.[29] NAP was also found effective
against mild cognitive impairment in phase II trials; however, it
failed to show efficacy for progressive supranuclear palsy in a phase
III trial.[30,31] A naturally occurring dipeptide,
carnosine, was reported as a very effective Aβ aggregation lowering
agent at 10 μM.[32] Another designed
dipeptide (d-Trp-Aib-OH) wasproven to be a promising inhibitor.[33] A peptide D3 exhibited high binding affinity
toward Aβ and directed the equilibrium toward nontoxic aggregates.[34] D3 and some of its derivatives have undergone
phase-II clinical trials and may be candidates for advanced clinical
trials.[35,36]Fragments Aβ31–42, Aβ38–42, and Aβ39–42 derived from the hydrophobic
C-terminus region were found to reduce the Aβ aggregation significantly.[37] A recent report investigated the inhibitory
effects of pentapeptides derived from the fragment Aβ38–42.[38] A number of peptides were discovered
to exhibit significant to complete inhibition of Aβ1–42 aggregation. In another study, N-methylation performed on the hexapeptide
fragment, Aβ32–37 was shown to afford efficient
inhibitors of Aβ aggregation.[39] By
exploring the same fragment Aβ32–37, a hexapeptide
was identified to completely abrogate Aβ toxicity at sub-micromolar
concentrations.[40]In continuation
of our studies on the design of new inhibitors
of Aβ fibrillation, herein, we describe the synthesis of new
peptides utilizing further the relatively less explored C-terminus
region of Aβ. Synthesis was followed by biological evaluation
of the new peptides for their potential activity against Aβ
fibrillation. Primary screening by MTT cell culture assay was followed
by the ThT fluorescence assay for the selected peptides. Further,
we performed secondary structural analysis by circular dichroism (CD)
spectroscopy and morphological examination of Aβ aggregates
by electron microscopy to confirm the inhibitory activity of the best
tetrapeptide 20. Amino acid scan on the tetrapeptide
fragment Aβ39–42 was performed, wherein we
independently replaced all four amino acid residues of the sequence
by their isosteric physico-chemically analogous counterparts. Using
solid-phase peptide synthesis (SPPS) protocol, 43 new peptides were
synthesized by Fmoc chemistry using automated peptide synthesizer.
A general route for the synthesis of tetrapeptides exemplified by
peptide 20 has been depicted in Scheme . To start with, Wang resin with preloaded
C-terminus amino acid was first swelled and the Fmoc group was removed
by 20% piperidine in dimethylformamide (DMF). Next, the resin with
the free amino group was coupled with the sequential amino acids required
to synthesize the desired peptide sequence, in the presence of the
coupling reagent O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate (TBTU) or 1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid
hexafluorophosphate (HATU) and base N,N-diisopropylethylamine (DIEA), in DMF for 2.5 h. Kaiser’s
test was performed to routinely monitor the coupling reaction. Once
the peptide with the desired sequence was obtained, the Fmoc group
at the N-terminus was removed with 20% piperidine, and peptide was
cleaved from the resin by using 95% trifluoroacetic acid (TFA) in
triisopropylsilane (TIPS) and water. The so-obtained crude peptides
were purified and analyzed for purity by using reverse-phase high-performance
liquid chromatography (HPLC).
The newly
synthesized tetrapeptides
were first tested for their Aβ aggregation inhibitory potential
by MTT cell culture assay. In the initial screening, performed to
evaluate the synthesized tetrapeptides against the self-assembly and
toxicity of Aβ peptides, a number of peptides were observed
to rescue PC-12 cells from the Aβ aggregation-related toxicity.
Considering the cell viability of untreated cells as 100%, only 70%
cells were found to be viable when treated with 2 μM of Aβ1–42. The tetrapeptide (1, Val-Val-Ile-Ala),
previously reported active against the aggregation of Aβ1–42 peptide was found to exhibit significant inhibition
activity in this study as well. When tetrapeptide 1 (20
μM) was co-incubated with Aβ1–42, about
88% cells were found to be viable, corresponding to about 60% inhibition
of Aβ toxicity. In the presence of four of the tetrapeptides, 18 (Val-d-Pro-Ile-Ala), 20 (Val-Aib-Ile-Ala), 24 (Aib-Aib-Ile-Ala), and 25 (Pro-Pro-Ile-Ala),
cell viabilities were significantly improved. The tetrapeptide 20 (20 μM) was found to completely rescue PC-12 cells
against Aβ1–42 toxicity, whereas in the presence
of peptides 18, 24, and 25 about
89, 92, and 97% cells were found to be viable at 20 μM concentration
(Table ). Complete
restoration of cell viability in the presence of peptide 20 indicates total inhibition of Aβ1–42 aggregation
and toxicity. Four other tetrapeptides, 16 (20 μM,
Val-d-Ile-Ile-Ala), 19 (20 μM, Val-Pro-Ile-Ala), 32 (2 μM, Val-Val-d-Pro-Ala), and 42 (20 μM, Val-Val-Ile-Ile), showed only slight improvements
in cell viabilities and showed inhibition of amyloid toxicity in the
range of only 20–40%. However, the peptide 20,
found to completely prevent Aβ1–42 aggregation,
did not show inhibition toward Aβ1–40 toxicity
for PC-12 cells (data not shown). The cell viabilities in the co-presence
of either of these peptides were no different from those where PC-12
cells were incubated with Aβ1–40 alone. Cell
viability data correspondingly converted to percentage Aβ1–42 inhibition values for the tested tetrapeptide analogues
have been compiled in Table .
Table 1
Cell Viabilities and Percentage Inhibition
of Aβ1–42 Toxicity by Tetrapeptidesa
test concentrations
peptides
cell viability
inhibition (%) of Aβ1–42 (2 μM)
no.
sequence
20 μM
10 μM
2 μM
20 μM
10 μM
2 μM
1
Val-Val-Ile-Ala
88.0
79.0
72.4
60.0
30.0
8.0
2
d-Val-Val-Ile-Ala
71.2
70.7
69.5
9.0
5.0
4.0
3
d-Ala-Val-Ile-Ala
70.0
70.0
70.0
0
0
0
4
d-Leu-Val-Ile-Ala
70.6
70.0
70.0
2.0
0
0
5
Leu-Val-Ile-Ala
70.0
70.0
70.0
0
0
0
6
d-Ile-Val-Ile-Ala
70.0
70.0
70.0
0
0
0
7
Ile-Val-Ile-Ala
70.6
70.9
70.0
2.0
3.0
0
8
d-Pro-Val-Ile-Ala
71.2
72.7
71.5
4.0
9.0
5.0
9
Pro-Val-Ile-Ala
70.0
70.0
70.0
0
0
0
10
Aib-Val-Ile-Ala
73.9
72.7
70.3
13.0
9.0
1.0
11
Nva-Val-Ile-Ala
70.0
70.0
70.0
0
0
0
12
Val-d-Val-Ile-Ala
70.0
70.0
70.0
0
0
0
13
Val-d-Ala-Ile-Ala
70.0
70.0
70.0
0
0
0
14
Val-d-Leu-Ile-Ala
70.3
70.0
71.5
1.0
0
5.0
15
Val-Leu-Ile-Ala
73.9
70.0
70.0
13.0
0
0
16
Val-d-Ile-Ile-Ala
78.1
76.0
76.0
27.0
20.0
20.0
17
Val-Ile-Ile-Ala
70.0
70.0
70.0
0
0
0
18
Val-d-Pro-Ile-Ala
89.2
76.0
72.1
64.0
20.0
7.0
19
Val-Pro-Ile-Ala
77.2
75.1
74.8
24.0
17.0
16.0
20
Val-Aib-Ile-Ala
100
76.6
70.0
100
22.0
0
21
Val-d-Phe-Ile-Ala
70.0
71.5
70.0
0
5.0
0
22
Val-Phe-Ile-Ala
70.0
69.5
69.2
8.0
4.0
3.0
23
Val-Nva-Ile-Ala
71.2
70.7
69.5
9.0
5.0
4.0
24
Aib-Aib-Ile-Ala
91.9
75.4
73.9
73.0
18.0
13.0
25
Pro-Pro-Ile-Ala
97.0
81.7
74.2
90.0
39.0
14.0
26
Val-Val-d-Ile-Ala
70.0
70.0
70.0
0
0
0
27
Val-Val-d-Ala-Ala
73.3
70.0
70.0
11.0
0
0
28
Val-Val-d-Leu-Ala
73.0
70.6
70.0
10.0
2.0
0
29
Val-Val-Leu-Ala
70.0
70.0
73.0
0
0
10.0
30
Val-Val-d-Val-Ala
70.0
70.0
70.0
0
0
0
31
Val-Val-Val-Ala
70.9
70.0
70.0
3.0
0
0
32
Val-Val-d-Pro-Ala
76.3
77.8
78.1
21.0
26.0
27.0
33
Val-Val-Pro-Ala
70.9
70.0
70.0
3.0
0
0
34
Val-Val-Aib-Ala
73.3
70.0
70.3
11.0
0
1.0
35
Val-Val-d-Phe-Ala
72.7
73.0
71.2
9.0
10.0
4.0
36
Val-Val-Phe-Ala
70.0
70.0
70.0
0
0
0
37
Val-Val-Nva-Ala
70.3
70.0
70.0
1.0
0
0
38
Val-Val-Nle-Ala
70.0
70.0
70.0
0
0
0
39
Val-Val-Ile-d-Ala
71.2
70.0
70.0
4.0
2.0
0
40
Val-Val-Ile-Val
72.7
73.9
71.2
9.0
13.0
4.0
41
Val-Val-Ile-Leu
70.0
70.0
70.0
0
0
0
42
Val-Val-Ile-Ile
78.7
78.1
77.2
29.0
27.0
24.0
43
Val-Val-Ile-d-Phe
71.2
70.0
70.0
4.0
0
0
44
Val-Val-Ile-Phe
70.0
70.0
70.0
0
0
0
control
100
Aβ1–42
70.0
The final concentration
ratios of
Aβ and test peptides were kept as 1:1, 1:5, and 1:10. Experiments
were performed in triplicates (n = 3). Blank ODs
were subtracted from each sample OD and the triplicate ODs were averaged.
In a subset of triplicate wells, SD values ranged 1.35–4.98.
Aib, aminoisobutyric acid; Nva, norvaline; Nle, norleucine.
The final concentration
ratios of
Aβ and test peptides were kept as 1:1, 1:5, and 1:10. Experiments
were performed in triplicates (n = 3). Blank ODs
were subtracted from each sample OD and the triplicate ODs were averaged.
In a subset of triplicate wells, SD values ranged 1.35–4.98.
Aib, aminoisobutyric acid; Nva, norvaline; Nle, norleucine.
Structure–Activity Relationship
On the basis
of the inhibitory activities shown by tetrapeptide derivatives in
the cell viability assay, a structure–activity relationship
was established. For the purpose of a start point, the inhibition
activity shown by peptide 1 (60%, 20 μM) was taken
as a reference. Residue Ile41 of the tetrapeptide (Val39-Val40-Ile41-Ala42) was
found to be crucial for its activity and substitutions by residues
such asd-Ile, d/l-Leu, d/l-Pro, d/l-Phe, d/l-Val,
aminoisobutyric acid (Aib), Nva, Nle, and d-Ala at this position
resulted in the loss of inhibitory activity. None of the peptides
designed by substitution at Ile41 showed more than 30%
inhibition of Aβ aggregation. Also, the substitution at the
Val39 by residues such asAib, Nva, d/l-Pro, d/l-Ile, d/l-Leu, d-Ala, and d-Val did not result into significant activity.
Substitutions at the next residue (Val40) by d-Pro (Val-d-Pro-Ile-Ala, 18) afforded a peptide
that showed considerable inhibition (64%) of Aβ fibrillation,
whereas the peptide obtained by substitution Val40 →
Aib (Val-Aib-Ile-Ala, 20) exhibited complete inhibition
of Aβ fibrillation. However, other replacements by d-Val, d/l-Leu, d/l-Pro, d/l-Phe, d/l-IleAib, Nva, and d-Ala at this position resulted in inactive peptides. The substitutions
at the C-terminus residue (Ala42) by Val, Leu, d/l-Phe, Ile, and d-Ala also did not yield any compound
showing significant inhibition toward Aβ polymerization. By
utilizing the known anti-β-sheet property of Aib and Pro residues,
two more peptides [Aib-Aib-Ile-Ala (24) and Pro-Pro-Ile-Ala
(25)] were designed and synthesized by replacing both
Val39 and Val40 amino acids of the peptide 1, simultaneously. These peptidesproved to be good inhibitors
(73% inhibition, 24) and (90% inhibition, 25) of amyloid aggregation. Because most of the positive substitutions
(active peptides) were obtained by replacement of residues in the
leadpeptide by Aib and Pro, such an analysis supports the widely
accepted β-sheet breaking capabilities of these residues. Specific
interaction of the best inhibitor tetrapeptide (20) with
the last two residues (Ile41 and Ala42) in Aβ1–42 is evidenced by its lack of activity against the
smaller aggregating peptide Aβ1–40.
Thioflavin
T (ThT) Fluorescence Study
Three best tetrapeptides, 20, 24, and 25, showing amyloid-β
toxicity inhibition in the range of 73–100%, in the cell viability
assay, were further studied by ThT fluorescence assay. Upon co-incubation
of tetrapeptides 20, 24, and 25 with the Aβ1–42 peptide, remarkable reduction
in the ThT fluorescence was observed. Graphical representation of
the relative ThT fluorescence, for the Aβ1–42 incubated alone, or plus the tetrapeptides is described in Figure .
Figure 1
Graphical demonstration
of the effects of tetrapeptides 20, 24,
and 25 on Aβ1–42 aggregation-mediated
ThT fluorescence. ThT dye alone is represented
as control while amyloid peptides incubated along with the dye are
shown as Aβ1–42. Aβ1–42 was taken as 2 μM and the tetrapeptides 20, 24, and 25 were kept in ratios of 1:1, 1:5, and
1:10 (Aβ/test peptides) in the final incubated solutions. Sequential
bars describe the co-incubated inhibitor peptides (indicated by numbers),
with the Aβ peptide and ThT dye. Error bars represent mean ±
SD (n = 3). Data were analyzed by one-way ANOVA test
followed by Dunnett’s multiple comparison test (ns—non
significant, $p < 0.05, #p < 0.01, *p < 0.001, vs
Aβ) using software (GraphPad Prism, ISI, San Diego, CA). Experiments
were performed in triplicates (n = 3). Blank values
were subtracted from that of sample fluorescence and triplicates were
averaged. In a subset of triplicate wells, SD values ranged 1.34–6.38.
Graphical demonstration
of the effects of tetrapeptides 20, 24,
and 25 on Aβ1–42 aggregation-mediated
ThT fluorescence. ThT dye alone is represented
as control while amyloid peptides incubated along with the dye are
shown as Aβ1–42. Aβ1–42 was taken as 2 μM and the tetrapeptides 20, 24, and 25 were kept in ratios of 1:1, 1:5, and
1:10 (Aβ/test peptides) in the final incubated solutions. Sequential
bars describe the co-incubated inhibitor peptides (indicated by numbers),
with the Aβ peptide and ThT dye. Error bars represent mean ±
SD (n = 3). Data were analyzed by one-way ANOVA test
followed by Dunnett’s multiple comparison test (ns—non
significant, $p < 0.05, #p < 0.01, *p < 0.001, vs
Aβ) using software (GraphPad Prism, ISI, San Diego, CA). Experiments
were performed in triplicates (n = 3). Blank values
were subtracted from that of sample fluorescence and triplicates were
averaged. In a subset of triplicate wells, SD values ranged 1.34–6.38.ThT dye alone (control) exhibited
relative fluorescence unit (RFU)
of 30%. In the presence of inhibitor tetrapeptides, RFU values did
not deviate much from control and were quite close to the dye alone
values. Such as in the presence of the peptide 20, RFU
value of 31.4% (20 μM) was observed. Likewise, tetrapeptides 24 and 25 co-incubated samples showed RFU values
of 42.6 and 38.4% at 20 μM, respectively. Diminution of ThT
fluorescence upon co-incubation of tetrapeptides 20, 24, and 25 with Aβ1–42 peptide supports the results of MTTassay for their inhibitory activity
toward the prevention of Aβ1–42 self-assembly.
However, similar to what was observed in the MTTassay, the tetrapeptides
did not show inhibitory activity against the Aβ1–40 peptide (data not shown). The RFU values similar to that of Aβ1–40 alone point toward the formation of Aβ aggregates
that indicates the inefficiency to inhibit the Aβ1–40 peptide aggregation.Tetrapeptide 20 (Val-Aib-Ile-Ala)
was further studied
in a time-dependent manner and ThT fluorescence was measured at regular
intervals for 7 days. ThT dye was incubated with aggregating peptide
Aβ1–42 alone or plus the inhibitor peptide 20 so as to result into final solution concentrations in the
ratio of 1:10 (Aβ/peptide 20). When the ThT dye
was incubated with Aβ1–42 alone, remarkable
enhancement in the fluorescence was observed indicating the presence
of large Aβ1–42 aggregates. After a slow rise
in fluorescence intensity for initial 12 h, ThT fluorescence showed
an exponential phase and reached saturation at about 120 h. Compared
with the Aβ1–42 alone, ThT fluorescence was
significantly reduced in the co-presence of the inhibitor peptide 20. Only 43.4% RFU was observed when measured at 12 h. The
subsequent measurements at various intervals showed even lesser values
of RFUs. At 24 h, 3 days, 5 days, and 7 days after co-incubation of
peptide 20 with Aβ1–42, relative
fluorescence values of 6, 4, 10.9, and 11.4% were observed, respectively
(Figure ). Because
ThT fluorescence is a direct indicator of the amyloid aggregation
state, the reduction in the fluorescence demonstrates the inhibitory
activity of tetrapeptide 20 toward Aβ1–42 aggregation. Thus, peptide 20 retained its inhibitory
effects on the Aβ1–42 aggregation even after
prolonged incubation. Hence, the results of the fluorescence assay
have been found in good agreement and correlated well with those obtained
in the MTT-based assay.
Figure 2
Time-dependent studied inhibitory effects of
tetrapeptide 20 on Aβ1–42 aggregation.
Red line
represents the Aβ1–42 aged alone, whereas
Aβ1–42 peptide co-incubated with peptide 20 is shown in green. The aggregating peptide Aβ1–42 (2 μM) and inhibitor peptide 20 (20 μM) were taken in the final solution concentrations in
the ratio of 1:10 (Aβ/peptide 20). Error bars represent
mean ± SD (n = 3). Data were analyzed by t-test ($p < 0.05, #p < 0.01, *p < 0.001, vs
Aβ1–42) using software GraphPad Prism, ISI,
San Diego, CA. Each experiment was performed in triplicates (n = 3) and triplicates were averaged. In a subset of triplicate
wells, SD values ranged 0.22–1.32.
Time-dependent studied inhibitory effects of
tetrapeptide 20 on Aβ1–42 aggregation.
Red line
represents the Aβ1–42 aged alone, whereas
Aβ1–42 peptide co-incubated with peptide 20 is shown in green. The aggregating peptide Aβ1–42 (2 μM) and inhibitor peptide 20 (20 μM) were taken in the final solution concentrations in
the ratio of 1:10 (Aβ/peptide 20). Error bars represent
mean ± SD (n = 3). Data were analyzed by t-test ($p < 0.05, #p < 0.01, *p < 0.001, vs
Aβ1–42) using software GraphPad Prism, ISI,
San Diego, CA. Each experiment was performed in triplicates (n = 3) and triplicates were averaged. In a subset of triplicate
wells, SD values ranged 0.22–1.32.
Circular Dichroism Spectroscopy
Under abnormal conditions,
Aβ peptides undergo conformational changes and aggregates in
the form of cross-β-sheet structure. Such a transition in the
conformation from a mixture of various secondary structures toward
β-sheet can be estimated quantitatively by using CD spectroscopy.[41] Tetrapeptide 20 (Val-Aib-Ile-Ala)
was studied for its inhibitory effects on the conformational transition
of Aβ1–42 peptide. Aβ and the inhibitor
tetrapeptide 20 were mixed in a ratio of 1:5 and incubated
for 12 h (t12h) before the secondary structure
analysis was performed. As shown in Figure , Aβ1–42 aged alone
for 12 h, shows an increase in the β-sheet content as evident
by the development of a broad negative peak at 217–218 nm.
Initial (t0h) secondary structure analysis
of Aβ1–42 yielded a mixture of 9.5% of β-sheet,
while being 61.8% in random coil and 28.7% β-turn forms. However,
at 12 h of incubation, β-sheet component was increased to 47.1%,
with the random coil reduced to 42.3%. To analyze secondary structure
of Aβ1–42 peptide in the presence of inhibitor
peptide 20, inhibitor spectrum was subtracted from that
of inhibitor-treated Aβ1–42.
Figure 3
Effects of peptide 20 on the conformational transition
of Aβ1–42 studied by CD spectroscopy. Aβ1–42 and the inhibitor tetrapeptide 20 were
mixed in a ratio of 1:5 and incubated for 12 h (t12h) before the secondary structure analysis was performed.
Shown in black and red are the CD spectra of Aβ1–42 at t0h and t12h, respectively. Blue line shows the CD spectra of Aβ1–42 peptide co-incubated with peptide 20 at 12 h.
Effects of peptide 20 on the conformational transition
of Aβ1–42 studied by CD spectroscopy. Aβ1–42 and the inhibitor tetrapeptide 20 were
mixed in a ratio of 1:5 and incubated for 12 h (t12h) before the secondary structure analysis was performed.
Shown in black and red are the CD spectra of Aβ1–42 at t0h and t12h, respectively. Blue line shows the CD spectra of Aβ1–42 peptide co-incubated with peptide 20 at 12 h.Upon co-incubation with peptide 20 for 12 h (t12h), Aβ1–42 exhibited
only 14.2% of β-sheet conformation while occupying 58.5% of
random coil form. As evident from Figure , the disappearance of minima at 217 nm in
the co-presence of inhibitor peptide 20 is a direct indication
of the inhibitory activity of the tetrapeptide 20.
Transmission Electron Microscopy (TEM)
Morphological
examination of the Aβ1–42 aggregates in the
co-presence of tetrapeptide 20 (Val-Aib-Ile-Ala) was
performed by TEM. Scanning TEM (STEM) was further utilized to investigate
shapes and appearance of Aβ1–42 assemblies.
Peptide 43 (Val-Val-Ile-d-Phe, inactive in MTTassay) was taken as a negative control. An extensive network of long,
cylindrical rod-like aggregates was observed when Aβ1–42 was aged alone. No fibril ends were observed in consecutive images
[Figure A, high-resolution
TEM (HRTEM) and Figure A, STEM]. While upon co-incubation with peptide 20,
no such aggregates were formed (Figure B, HRTEM and Figure B, STEM). Only small irregular sized particles were
detected. Peptide 20 also did not exhibit any self-aggregation
when incubated alone under similar conditions (Figure C, HRTEM and Figure C, STEM). TEM images of peptide 20 alone showed only scarcely spread small structures.
Figure 4
Visual investigation
of the aggregation of Aβ1–42 peptide in the
presence of tetrapeptides 20 and 43 by HRTEM.
Aβ1–42 and tetrapeptides 20 and 43 were mixed for a concentration ratio
of 1:5 (Aβ1–42/test peptides) and incubated
at 37 °C for 72 h. Aβ1–42 was incubated (A) alone,
(B) with inhibitor peptide 20, and (D) with the inactive
peptide 43. Peptides 20 and 43 incubated alone are shown in images (C,E), respectively. The scale
bar shows 0.2 μm.
Figure 5
Visual investigation of the aggregation of Aβ1–42 peptide in the presence of tetrapeptides 20 and 43 by STEM. Aβ1–42 and tetrapeptides 20 and 43 were mixed for a concentration ratio
of 1:5 (Aβ1–42/test peptides) and incubated
at 37 °C for 72 h. Aβ1–42 was incubated,
(A) alone, (B) with inhibitor peptide 20, and (D) with
the inactive peptide 43. Peptides 20 and 43 incubated alone are shown in images (C,E), respectively.
The scale bar shows 500 nm.
Visual investigation
of the aggregation of Aβ1–42 peptide in the
presence of tetrapeptides 20 and 43 by HRTEM.
Aβ1–42 and tetrapeptides 20 and 43 were mixed for a concentration ratio
of 1:5 (Aβ1–42/test peptides) and incubated
at 37 °C for 72 h. Aβ1–42 was incubated (A) alone,
(B) with inhibitor peptide 20, and (D) with the inactive
peptide 43. Peptides 20 and 43 incubated alone are shown in images (C,E), respectively. The scale
bar shows 0.2 μm.Visual investigation of the aggregation of Aβ1–42 peptide in the presence of tetrapeptides 20 and 43 by STEM. Aβ1–42 and tetrapeptides 20 and 43 were mixed for a concentration ratio
of 1:5 (Aβ1–42/test peptides) and incubated
at 37 °C for 72 h. Aβ1–42 was incubated,
(A) alone, (B) with inhibitor peptide 20, and (D) with
the inactive peptide 43. Peptides 20 and 43 incubated alone are shown in images (C,E), respectively.
The scale bar shows 500 nm.However, when Aβ1–42 peptide was
incubated
with the inactive tetrapeptide 43, small truncated aggregates
were found in bunches (Figure D, HRTEM and Figure D, STEM). Peptide 43, when aged alone was also
seen to form large clusters of amorphous aggregates (Figure E, HRTEM and Figure E, STEM). Therefore, electron
microscopy study further corroborates the inhibitory activity of the
tetrapeptide 20.
Cytotoxicity Study
Because the most active tetrapeptide 20 comprise all
hydrophobic amino acid residues, whether it
self-aggregates becomes worth-testing. Following a similar procedure
as for the activity study, it was found that the inhibitor tetrapeptide 20 did not exert cytotoxic effects on the PC-12 cells at the
highest tested concentration (20 μM). Cell samples treated with
inhibitor peptide 20 showed viability as 97.7% (SD: 4.4).
Conclusions
In view of the specific binding of peptide fragments
for the parent
peptide, amino acid scan was performed by means of independent amino
acid replacement on the tetrapeptide fragment of Aβ1–42 and 43 new peptides were synthesized. Newly synthesized peptides
consisted of both l- as well as d-isomers of the
newly substituted amino acid residues. New tetrapeptide analogues
were initially screened for their inhibition potential against the
aggregating Aβ peptides by the MTTassay. ThT fluorescence experiments
carried out for the best peptides well supported MTT results. CD spectral
analysis and electron microscopy performed for one of the best inhibitor
peptide further confirmed the observations. Total deterrence of Aβ
aggregation along with noncytotoxic profiles makes the specific tetrapeptide 20 discovered herein as a promising lead for further development
against AD.
Experimental Section
General Chemistry
All of the chemicals
used for peptide
synthesis were purchased from Sigma-Aldrich, Missouri, U.S.A,. and
Chem-Impex International, Illinois, U.S.A., and used without further
purification, unless otherwise stated. The solvents used were of analytical
grade and used without further purification. Peptides were synthesized
on a fully automated peptide synthesizer (AAPPTec Focus XC) by SPPS
protocol using Fmoc chemistry on 0.1 mM scale. Wang resin, used as
solid support, was preloaded with the C-terminus amino acid. 1H and 13C NMR spectra were recorded on a Bruker
AVANCE III 400 MHz instrument using CD3OD as the solvent
and tetramethylsilaneas the internal standard. Proton and carbon
chemical shifts are expressed in parts per million (ppm, δ scale)
and were referenced to NMR solvent CD3OD, δ 3.31,
49.0 ppm. The following abbreviations were used to describe peak patterns
when appropriate: br = broad, s = singlet, d = doublet, t = triplet,
q = quadruplet, m = multiplet. Coupling constants (J) were reported in hertz unit (Hz). High-resolution mass spectroscopy
(HRMS) spectra were obtained with MAXIS-Bruker using the electrospray
ionization time-of-flight (ESI-TOF) method.
General Method for the
Synthesis of Peptides
After
swelling the resin in DMF for 15 min in a sintered glass-bottom reaction
vessel, Fmoc group was delinked from the C-terminus amino acid residue
preloaded on the Wang resin, by using 20% piperidine in DMF for 15
min (2 × 10 mL). Afterward, the next residue followed by its
in situ activation by treating with TBTU (3.0 equiv, 0.5 M in DMF)
and DIEA (3.0 equiv, 1.0 M in DMF) was coupled with the resin bound
C-terminus residue for 2.5 h at ambient temperature by mechanical
shaking. HATU instead of TBTU was used as a coupling reagent to couple
Aib. Kaiser’s test was routinely utilized to monitor coupling
reactions. Fmoc removal and subsequent coupling cycles with the next
amino acid residues were repeated in succession to achieve the desired
tetrapeptides. Tetrapeptides were first reacted with 20% piperdine
for 15 min to remove N-terminus Fmoc groups. Peptides were then freed
from resin and other protecting groups by magnetically stirring the
resin-bound peptides in a cocktail of TFA/TIPS/H2O (95:3:2,
15 mL) for 2.5 h at ambient temperature. The solution was filtered
and filtrate was concentrated to 0.5 mL. Addition of cold diethyl
ether to the concentrate afforded the crude peptides. Crude peptides
dissolved in a 1:1 mixture of H2O–CH3OH (v/v) were loaded on a preparative reversed-phase (RP)-HPLC (Shimadzu
Prominence LC-8A model using a Phenomenex column, LC-18, 250 ×
21.2 mm, 10 μm), operating at 215 nm. TFA (0.1%) in CH3CN (A) and 0.1% TFA in water (B) were used as mobile phase. A linear
gradient of 10–60% of mobile phase A was run for 40 min at
a flow rate of 21 mL/min. The desired fractions were pooled together
and concentrated. Peptides were lyophilized by dissolving in 80% aqueous
acetic acid. Purity check of the peptides was performed by analytical
RP-HPLC (Shimadzu Prominence RP-HPLC system using a Phenomenex column,
LC-18, 250 × 4.6 mm, 5 μm) by running a gradient run of
40 min, with a linear increase (10–90%) in mobile phase A.
The flow rate was kept as 1.0 mL/min and operated at wavelength of
215 nm. The analysis showed the peptide purity in the range of 95–100%.
The peptides were characterized and ascertained for identity by HRMS
taken on a Maxis Bruker spectrometer and nuclear magnetic resonance
spectroscopy using AVANCE III 400 Bruker (400 MHz).
[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]bromide
(MTT) reduction in the mitochondria of living cells results into purple-colored
formazan crystals that can be measured spectrophotometrically. The
observed optical density (OD) is directly proportional to the number
of live cells, hence, of utmost importance in the evaluation of cellular
viability in drug screening and neuronal cytotoxicity.[43] PC-12 cells were purchased from National Center
for Cell Science (NCCS), Pune, India. Aβ peptides were purchased
from AnaSpec Inc., USA. Horse serum was purchased from HiMedia Laboratories,
India. 96-well, flat-bottom microtiter plates (Costar), Corning tissue-culture
treated dishes, F-12K growth media, sodium bicarbonate, MTT, penicillin/streptomycin
and fetal bovine serum (FBS, heat inactivated) were purchased from
Sigma-Aldrich Chemicals, Saint Louis, MO, USA. Phosphate-buffered
saline (PBS, pH 7.4) was purchased from Gibco (Life Technologies).
HPLC grade dimethyl sulfoxide (DMSO) was purchased from Alfa Aesar.
All of the solutions were filtered via 0.2 mM syringe filters for
sterilization. OD (OD570) measurements were performed using
a microtiter plate reader (VERSA max tunable; Molecular Devices, Sunnyvale,
CA). PC-12 cells were cultured in F-12K growth media supplemented
with 5% FBS, 10% horse serum, and 1% penicillin/streptomycin in a
humidified atmosphere with 5% CO2 at 37 °C. Cells
were grown in tissue-culture-treated dishes and used when 70% confluent.
Just before the experiments, the media was replaced by F-12K containing
10% FBS and 1% penicillin/streptomycin. To ensure the reproducibility
in monitoring Aβ self-assembly, Aβ peptides were monomerized
by following Zagorski’s protocol as explained elsewhere.[40,42] Briefly, three cycles of dissolution, sonication in TFA (predistilled),
and drying under vacuum was followed by HFIP treatment. Aβ peptides
were dissolved in HFIP, sonicated, and dried under nitrogen. HFIP
traces were removed under high vacuum (0.5 mm Hg) for 2 h. Peptides
were finally dissolved in 1 mL of HFIP and aliquoted as 100 μg
stocks. Just before the experiments, an aliquot of monomerized Aβ
peptide was diluted in 20 mM NaOH to a concentration of 200 μM.
Aβ peptides were further diluted to 20 μM by adding 10
mM sodium phosphate buffer (SPB, pH 7.4). PC-12 cells were seeded
at a rate of 17 000 cells per well per 80 μL, and incubated
overnight. Test peptides dissolved in DMSOas 5 mM stock solutions
were diluted to concentrations of 200, 100, and 20 μM, in PBS,
immediately before the experiments. Next day, 10 μL of Aβ
was taken in each well with and without the test peptides (10 μL)
and incubated for 6 h. The final concentration ratios of Aβ
and test peptides were kept as 1:1, 1:5, and 1:10. After 6 h, MTT
(20 μL, 5 mg/mL in PBS) wasadded and further incubated for
4 h. The plates were centrifuged for 10 min at 4 °C. After removing
the supernatant carefully, DMSO (200 μL, per well) wasadded.
The suspension was well-mixed and OD570 were measured.
Untreated cell samples were taken as control. Setting the ODs of untreated
cell samples to 1, the cell viability was considered as 100. Corresponding
percentage cell viabilities were calculated for the cell samples treated
with Aβ alone or plus the test peptides. Using the formula,
100 × [OD570 (test peptide with Aβ1–42) – OD570 (Aβ1–42)]/[OD570 (control) – OD570 (Aβ1–42)], percentage inhibition of Aβ toxicity by each tetrapeptide
was calculated. Statistical analysis was performed by the one-way
ANOVA test followed by Dunnett’s multiple comparison test ($p < 0.05, #p < 0.01, *p < 0.001, vs Aβ) using software
GraphPad Prism, ISI, San Diego, CA. p Values less
than 0.05 were considered as significant.
Cytotoxicity Assay
PC-12 cells were seeded in 96-well
plates, @ 17 000 cells per well per 90 μL, and incubated
overnight. Next day, 10 μL of 200 μM (DMSO/PBS in 1:10
ratio) inhibitor tetrapeptide 20 wasadded to make a
final nominal concentration of 20 μM. After 6 h of incubation,
20 μL of MTT (5 mg/mL in PBS) wasadded and incubated further
for 4 h. The plate was centrifuged for 10 min at 4 °C. Next,
the supernatant was carefully discarded and DMSO wasadded (200 μL,
per well). The suspension was mixed well and ODs (OD570) were measured using a microtiter plate reader. Experiments were
performed in triplicates (n = 3) and ODs were averaged.
OD of untreated cell samples was set to 1 and background ODs were
subtracted from each sample OD. MTT reduction (%) in the presence
of test peptides was determined by comparing the OD570 of
each test sample to the OD570 of the control.
ThT Fluorescence
Assay
Because ThT shows enhanced fluorescence
signal upon binding to protein aggregates, it is quite useful in the
quantitative measurement of the presence of Aβ aggregates.[44] ThT and black, clear-bottom, 96-well plates
were purchased from Sigma-Aldrich Chemicals, Saint Louis, MO, USA.
Presterilization was effected via filtration using 0.2 μm syringe
filters. Fluorescence measurements were performed on Tecan M200 plate
reader. Aβ1–42 and Aβ1–40 were taken as 2 and 10 μM, respectively, and the tetrapeptides 20, 24, and 25 were taken in ratios
of 1:1, 1:5, and 1:10 (Aβ/test peptides). The incubation periods
for Aβ1–42 and Aβ1–40 were 24 and 72 h, respectively. Aβ1–42 dissolved
in 20 mM NaOH (400 μM) was further diluted to 20 μM in
SPB (pH 7.4). ThT (2.5 μM) dissolved in glycine–NaOH
buffer (pH 8.5) wasadded (120 μL) in each well of a black,
clear-bottom 96-well plate. Tetrapeptides 20, 24, and 25 (5 mM in DMSO) were diluted in PBS to 200,
100, and 20 μM set of concentrations. Aβ1–42 (15 μL) wasadded to each well followed by test peptides (15
μL) to obtain a ratio of 1:1, 1:5, and 1:10. With mechanical
shaking at 200 rpm, the plate was incubated at 37 °C for 24 h.
After incubation, the plates were read at wavelengths of 445 nm (λex) and 485 nm (λem). The enhancement in the
ThT fluorescence shown by Aβ1–42 comparative
to blank was taken as 100%, and fluorescence values were calculated
for inhibitor peptide samples co-incubated with Aβ1–42. Inhibition activity was calculated by the relative percent enhancement
in the fluorescence of the amyloid-bound ThT fluorescence of the target–inhibitor
mixture compared with that in the absence of the inhibitor. Table
S1 (Supporting Information) shows the inhibition
(%) of aggregation in the presence of the inhibitor tetrapeptides
against Aβ1–42. In the time-dependent study
performed on the tetrapeptide 20, the increase in ThT
fluorescence upon incubation with Aβ1–42 relative
to ThT alone (control) was taken as 100% and the RFU values were normalized
for the co-incubated inhibitor peptide 20 with the Aβ1–42. The fluorescence observed in the blank wells was
subtracted from that of test samples. Statistical analysis was performed
by a one-way ANOVA test followed by Dunnett’s multiple comparison
test ($p < 0.05, #p < 0.01, *p < 0.001, vs Aβ)
using software GraphPad Prism, ISI, San Diego, CA. p Values less than 0.05 were considered as significant.
Circular Dichroism
Spectroscopy
Calibration of the
instrument, JASCO, J-815 CD spectrometer was performed by using freshly
prepared (0.6 w/v %) ammonium salt of (+)-camphor-10-sulfonic acid.
CD spectra were recorded using a 1 mm quartz cell in the wavelength
range of 190–260 nm at 37 °C. Data points were collected
at a speed of 50 nm per min with 0.2 nm intervals, a response time
of 1 s and a band width of 2 nm. The data are presented as an average
of 3 scans recorded in succession. Aβ1–42 predissolved
in 20 mM NaOH (200 μM) was diluted in SPB to 20 μM. Inhibitor
peptide 20 (1.0 mM in TFE) was made up to 100 μM
by diluting in SPB. To obtain a concentration ratio of 1:5 (Aβ1–42/20), 500 μL of each of Aβ1–42 and peptide 20 were mixed and incubated
for 12 h (t12h) at 37 °C. To determine
final CD spectra, a buffer blank spectrum was subtracted from that
acquired for the samples. Noise was reduced by smoothing the far-UV
CD spectra. The direct CD measurements (θ, in mdeg) were converted
to molar ellipticity, using [θ] = θ/(10·C·l), where C and l denotes the molar concentration (mol/L) and path length, respectively.
The molar ellipticity [θ] is expressed in units of deg cm2 dmol–1. Data were average of at least three
scans recorded in parallel for each run sample. Spectra manager II
was used for deconvolution of CD spectra and various forms of secondary
structures (%) were estimated.
Transmission Electron Microscopy
HRTEM and STEM investigations
were performed using FEI Tecnai (G2 F20) transmission electron microscope
operating at 120 keV. Glutaraldehyde (EM grade) and uranyl acetate
were purchased from Sigma-Aldrich chemicals, Saint Louis, MO, USA.
Carbon-coated copper grids (200 mesh) were purchased from Electron
Microscopy Sciences. An aliquot of Aβ1–42 peptide
(500 μM in 20 mM NaOH) was diluted to 50 μM in 10 mM SPB
(pH 7.4). Stock solutions of tetrapeptides (5 mM in DMSO) 20 and 43 (negative control), were diluted to a final
concentration of 250 μM in SPB. Equal volumes (25 μL)
of Aβ1–42 and tetrapeptides 20 and 43 were mixed for a concentration ratio of 1:5
(Aβ1–42/test peptides) and incubated at 37
°C for 72 h. After the incubation period, a droplet of the sample
was fixed on the grid by using 0.5% of glutaraldehyde solution. After
washing by ultrapure water, grids were stained negatively by 2% uranyl
acetate. The samples were air-dried and examined. Aβ1–42 alone plus the buffers in similar concentration was considered as
a control. For unbiased classification of fibril morphology and abundance,
each EM grid was investigated at various (>10) positions.
Scheme 1
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5