L-Dopa and dopamine conjugated naphthalenediimides modulate amyloid β toxicity

Article abstract of DOI:10.1039/c8ob01691g

The process of protein misfolding and aggregation to form neurotoxic species is strongly implicated in most of the neurodegenerative disorders. In particular, amyloid beta (Aβ) misfolding and aggregation is central to pathophysiological processes of Alzhe

Full text of DOI:10.1039/c8ob01691g

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DOI: 10.1039/C8OB01691G.
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L-Dopa and dopamine conjugated naphthalenediimides modulate
amyloid β toxicity
Received 00th January 20xx,
Accepted 00th January 20xx
Madhu Ramesh,a,† Pandeeswar Makam,a,† Chandrashekhar Voshavar,a,† Harshavardhan Khare,b
Kolla Rajasekhar,a Suryanarayanarao Ramakumar,b Thimmaiah Govindaraju*a
DOI: 10.1039/x0xx00000x
The process of protein misfolding and aggregation to form neurotoxic species is strongly implicated in most of the
neurodegenerative disorders. In particular, amyloid beta (Aβ) misfolding and aggregation is central to pathophysiological
processes of Alzheimer’s disease. Development of aggregation modulators has enormous implications in the discovery of
effective therapeutic agents for Alzheimer’s disease. Herein, we report the design and synthesis of series of natural amino
acids, L-dopa and dopamine appended derivatives of naphthalenediimide (NDI) to identify efficient aggregation
modulators. Furthermore, the molecular docking studies revealed the possible binding sites and binding mode of NDI-
conjugates to Aβ aggregates. Among the designed NDI-conjugates, L-dopa and dopamine derivatives (NLD and NDP,
respectively) showed excellent aggregation modulation efficiency (inhibition and dissolution), as shown by the thioflavin T
(ThT) binding assays, dot blot analysis and in cellulo studies. The docking results from in silico study are in good agreement
with the experimental data. In addition to significant modulation efficiency towards Aβ aggregation, NLD and NDP possess
antioxidant activity conducive to develop disease-modifying therapeutic agents for the treatment of Alzheimer’s disease.
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hypotheses centred around abnormal increase in the cerebral
senile plaques.7 The senile plaques predominantly consist of
Aβ42 peptide and is considered to play pivotal role in causing
Introduction
Alzheimer's disease (AD) is the most common
neurodegenerative disorder worldwide, described by the
progressive impairment of cognitive abilities leading to
dementia and death.1 The prevalence of AD increases with age
and severely effects population over the age of 60 years.
However, approximately 10-12% of population with the
disease have early onset AD which often appears at the young
age of 40-50.2 It was estimated that 35.6 million people were
suffering from dementia or related disorders worldwide in
2010 and projected to raise up to 70 and 135 million in 2030
and 2050, respectively. Neurodegeneration in hippocampus
and cerebral cortex of the brain caused by extracellular senile
plaques (SP) and intracellular neurofibrillary tangles (NFTs) is
the cardinal feature of Alzheimer’s disease.3,4 The protein
cleavage, misfolding, aggregation and depositions of the
aforementioned aggregates trigger an array of pathological
events ultimately leading to multifaceted neurotoxicity during
AD progression.5,6 Several hypotheses are proposed to
understand the pathophysiology of AD and most of these
multifaceted neuronal toxicity in the AD brain.8 The degree of
cognitive impairment in AD patients has been found to
correlate well with Aβ accumulation in the brain.9,10 Although
few drugs have been approved in the past few decades for the
treatment of AD condition, these drugs provide only
symptomatic relief and do not prevent the disease
progression.11 The mechanism of action of these drugs is
through modulation of signaling pathways (modulate
concentration levels of specific neurotransmitters in the brain)
to temporarily improve the memory and hence do not act on
the core pathology of AD. As a consequence, currently there is
an unmet need for the development of disease-modifying
therapeutic agents for the treatment of AD. In principle,
modulation of aggregation pathway is an attractive and
promising approach to develop disease-modifying therapeutic
agents.4 In addition, design of small molecules that can target
various pathological factors (multifunctional modulators)
associated with the multifactorial disease processes is
desirable.3,12-14 There are several approaches to modulate AD
pathology that include development of effective modulators of
Aβ aggregation, antioxidants and metal chelators among
others.15-17 Despite the significant progress in the AD research,
there are no effective approaches in designing multifunctional
Aβ modulators.
Several polyphenolic compounds such as epigallocatechin
gallate (EGCG), curcumin, resveratrol, ferulic acid and
hypericin have shown disrupting effect on the Aβ
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Targeting the hydrophobic pockets of Aβ aggregates through
hydrophobic moieties has been the key strategy for developing
aggregation modulators. This strategy prompted us to design
series of compounds with variable chemical functional groups
around a central hydrophobic aromatic core that can modulate
Aβ aggregation. We selected naphthalene aromatic core of
NDI as central hydrophobic moiety, to which chosen functional
groups were conjugated (Fig. 1A). NDIs are thermally stable,
planar π-electron deficient with well-defined redox and optical
properties and as a consequence finds a wide range of
applications from materials to biomedicine.26,27 Analogous to
aforementioned natural aromatic modulators, the planar and
compact aromatic skeleton of NDI in the designed compounds
is envisioned to modulate Aβ aggregation by interacting in the
hydrophobic pockets of Aβ polymorphic species. Further, we
assumed that the covalent functionalisation of NDI at imide
position with amino acids having variable
α-substituents and
phenolic moieties can potentially yield active compounds with
the ability to modulate Aβ aggregation process through
various non-covalent interactions and armed with intrinsic
antioxidant property. Accordingly, we conjugated NDI with
various aliphatic and aromatic natural amino acids (glycine,
alanine, tryptophan, phenylalanine and tyrosine in GNG
, ANA,
WNW, FNF and YNY, respectively). To introduce antioxidant
property, NDI was conjugated with special amino acid L-dopa
and dopamine as in NLD and NDP, respectively. Dopamine is a
well-known neurotransmitter, which is structurally similar to L-
dopa but lacks carboxylic acid group. Briefly, 1,4,5,8-
naphthalenetetracarboxylic dianhydride was dispersed in
dimethylformamide followed by the addition of triethylamine
and amino acids under reflux conditions. The product was
precipitated by acidifying the concentrated reaction mixture
by the addition of 1N HCl. The precipitate was collected by
filtration, washed with excess of distilled water and dried
under vacuo to obtain the natural amino acids, L-dopa and
Fig. 1 (A) Synthetic scheme and chemical structure of NDI modulators. (B) Schematic
representation for modulation of Aβ toxicity by NDI modulators.
aggregation.18 These natural compounds are known to perturb
the hydrophobic forces responsible for the formation of
amyloid aggregates.13,19-24 In addition, the polyphenolic
compounds exhibit excellent antioxidant property, a desirable
attribute necessary to modulate excessive reactive oxygen
species (ROS), oxidative stress and biomolecule damage.16, 17, 25
In this article, we report the design and synthesis of amino
acids, L-dopa and dopamine functionalised NDIs and the
detailed study to identify potential multifunctional modulator
of amyloid toxicity under in vitro conditions. Various
biophysical, in vitro and cell-based studies revealed that the L-
dopa (NLD) and dopamine (NDP) conjugated NDIs effectively
modulate Aβ42-aggregation and quench ROS, as a result these
modulators rescue PC12 cells from multifaceted amyloid
toxicity. Furthermore, the in vitro experimental results are
supported by the molecular docking studies which revealed
the interaction of NLD and NDP in KLVFFA, IIGLM regions of
Aβ42 with high binding affinities. Overall, these results clearly
indicate the potential of NLD and NDP to effectively modulate
Aβ42 aggregation, oxidative stress and rescue PC12 cells from
amyloid toxicity.
dopamine conjugated NDIs (ANA, GNG, YNY, WNW, FNF and
NLD) in good to excellent yields (70-90%) without the need of
any further purification. For NDP, acidification step with HCl
was excluded, the reaction mixture after DMF removal was
added with water and directly filtered using buchner funnel.
The precipitate was washed with copious amounts of water
and dried under vacuo to obtain NDP in quantitative yield. All
the compounds were characterised by NMR and high
resolution mass spectrometry (HRMS) analysis.
Modulation of Aβ11-25 aggregation
The NDI conjugates were studied for inhibition and dissolution
efficacies against Aβ11-25 aggregation by ThT assay. Aβ11-25
is a short hydrophobic fragment derived from Aβ42, which on
incubation in PBS buffer (10 mM, pH 7.4) at 37 °C attains β-
sheet conformation and form fibrillary aggregates similar to
full length peptide (Aβ42). For the inhibition experiment, 200
μM of Aβ11-25 was incubated without and with NDI
conjugates at 1:1 ratio for 48 h. ThT fluorescence assay
showed an intense fluorescence band at 482 nm for Aβ11-25
(control) indicating the formation of amyloid fibrils (Fig. S1). In
contrast, Aβ11-25 incubated independently with GNG and
Results and discussion
Design and synthesis of NDI-conjugates
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ANA showed slight decrease in fluorescence of ~ 44% and ~
39% respectively, while compounds FNF (~85%) and YNY
(~86%) caused five times decrease in the ThT fluorescence
intensity compared to Aβ11-25 alone. Remarkably, NLD and
NDP showed drastic decrease in fluorescence of 98% and 96%,
respectively. (Fig. S1). The fluorescence intensity of ThT is
directly correlated to the extent of amyloid aggregation and
the observed loss of fluorescence in the presence of NLD and
NDP is an indicative of inhibition of Aβ11-25 aggregation. Next,
we performed dissolution of pre-formed Aβ11-25 fibrils in the
presence of NDI conjugates. The Aβ11-25 pre-formed fibrils
were incubated with NDI conjugates for 48 h at 37 ⁰C and the
change in ThT fluorescence was measured in comparison with
untreated Aβ11-25 (Fig. S1). Interestingly, NLD and NDP
treated samples exhibited significantly low fluorescence
emission which is attributed to efficient dissolution of pre-
formed Aβ11-25 fibrils. FNF and YNY showed ~5 and ~3 fold
decrease in ThT fluorescence indicating the moderate
dissolution efficiency (Fig. S1). ANA did not show any
dissolution activity of pre-formed fibrils as indicated by the
unperturbed intense fluorescence band at 482 nm. Based on
this preliminary screening for dissolution of Aβ11-25
aggregates with different NDI conjugates, we performed time-
and concentration-dependent studies with most effective NDI
modulators (Fig. S2). In these experiments, each compound in
selected concentrations (10, 25 and 50 μM) was incubated
with pre-formed fibrils prepared using Aβ11-25 fragment for 0,
1, 2, 3 and 6 days. The data from the ThT fluorescence
measurements showed NLD and NDP are the most effective
aggregation modulators and the dissolution efficiency towards
Fig.
2
Inhibition and dissolution of Aβ42 aggregates by NDI modulators (ANA
,
FNF
,
WNW, NLD and NDP). (A) Aβ42 alone or with NDI modulators were
incubated for 24 h at 20 μM concentration at 1:1 ratio in PBS. (B) Dot blot
analysis of Aβ42 (10 μM) incubated with NDI modulators (10 μM). (C)
Quantification of dot blot. (D) Preformed Aβ42 aggregate (20 μM) was incubated
with NDI modulators at 20 μM (at 1:1 molar ratio) for 48 h for dissolution assay.
Values are the normalized maximal fluorescence intensity at 482 nm compared
to that of the control (Aβ42 without NDI modulators).
efficiency of 40%, 50% and 60%, respectively. This data
showed effective inhibitory effect of NLD and NDP towards
Aβ42 aggregation and these findings are consistent with the
ThT assay. Next, we performed concentration dependent
inhibition of Aβ42 (50 µM) aggregation using NLD and NDP at
1:1, 1:2 and 1:5 ratio of Aβ42 and NLD/NDP (Fig. S4). Both NLD
and NDP showed good inhibition efficiency of 80% and 85%,
respectively at 1:1 ratio. The percentage of inhibition efficiency
reached >90% with increase in the ratio of NDI-modulator.
Further, we analysed the Aβ42 aggregation in the absence and
presence of lead NDI modulators (NLD and NDP) through
transmission electron microscopy (TEM) analysis. The TEM
data showed that both NDP and NLD effectively prevent the
formation of Aβ42 fibrillar aggregates compared to control
(Aβ42 formed fully grown aggregates in the absence of
modulators) (Fig. S3).
Aβ11-25 fibrils was found to be in the order of NLD
WNW FNF YNY
Modulation of Aβ42 aggregation
> NDP >
>
>
.
The promising results from the preliminary screening of NDI
conjugates in modulating the Aβ11-25 aggregation encouraged
us to evaluate their potential against Aβ42 aggregation. We
performed a systematic study with active NDI modulators
(NLD, NDP, WNW and FNF) along with inactive ANA (Fig. 2). In
this experiment, 50 μM of Aβ42 in PBS (10 mM, pH 7.4) was
incubated alone or with 50 μM of NDI conjugates in the
presence of ThT for 24 h at 37 °C (Figure 2A). We found that
compounds NLD and NDP showed highest inhibition (~90%
and ~91% respectively) activities followed by WNW and FNF
(55% and 45% respectively), while ANA did not show
significant inhibition. Further, dot blot assay was performed to
confirm the inhibition efficiency of NDI modulators observed in
ThT assay. Aβ42 (10 µM) was incubated independently with 10
Next, we evaluated the ability of NLD, NDP, WNW and FNF
to dissolve the pre-formed Aβ42 fibrils by ThT fluorescence
assay. In this assay, both NLD and NDP showed maximum
dissolution ability of ~80%, while WNW and FNF exhibited
moderate dissolution efficiency (~50% and 30%, respectively)
of pre-formed fibrils (Fig. 2D). These findings are in good
agreement with corresponding dissolution profiles obtained
for Aβ11-25 aggregates. ANA did not show any significant
effect on the fibrils, as the ThT fluorescence intensity remained
unchanged and comparable to that of untreated Aβ42 fibrils.
In order to understand the effect of concentration on
dissolution ability, we performed concentration-dependent
study with NLD and NDP (Fig. S4). The dissolution effects of
NLD and NDP were found to be similar to the data from
concentration-dependent inhibition of Aβ42 aggregation (50
µM of ANA, FNF, WNW, NLD and NDP for 48 h at 37 °C. These
samples were blotted on PVDF membrane and treated with OC
primary antibody (binds to Aβ42 fibrillar aggregates) followed
by the fluorescent secondary antibody (labelled with Alexa
fluor-488).28 The fluorescent intensity of Aβ42 treated with
NDI-modulators was quantified in comparison to control
(untreated Aβ42). This data revealed that ANA and FNF did not
show any significant inhibition of Aβ42 aggregation (Figure 2B
and C). Whereas, WNW, NLD and NDP showed inhibition
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μM) (Fig. S4). NLD showed relatively superior dissolution effect of NDI conjugates on docking to different regions of Aβ fibrils
with increasing concentration (~88% at 1:1 ratio and ~93% at which is in agreement with the in vitro activity of the NDI
1:5 ratio) compared to NDP which exhibited dissolution modulators. In order to perform an unbiased search for
Fig. 3 Docking scores for two binding regions (A). (B) Ligand interaction analysis of docked ligands to KLVFFA and IIGLM regions. KLVFFA region shows docked poses of all 6 ligands,
whereas, IIGLM region does not show significant docking of GNG and ANA. (Figures generated using Ligplot+ Software). (C) XP Glide docking scores for the 6 ligands. (D) IFD
docking scores for the 6 ligands. (C) and (D) show distinctly better docking scores of NLD for both KLVFFA and IIGLM regions
efficiency of ~85% and ~95% at treatment ratios of 1:1 and 1:5, possible binding sites for NDI conjugates on the Aβ surface, we
respectively. Overall, the results from inhibition and adopted computational blind docking strategy followed by
dissolution experiments confirmed that NLD and NDP are the extra-precision and induced fit docking for selected sites (see
effective modulators of Aβ42 aggregation with former being ESI for detailed blind docking protocol). We used Glide
marginally superior over the later.
program in Schrodinger Software Suite32-34 for all the docking
studies. In contrast to usual docking studies where the binding
site is known, blind docking is an iterative method designed to
uniformly scan the entire surface of the receptor. We
automated this procedure through in-house written python
scripts that generate an equi-spaced grid over the surface of
the receptor and test the docking performance systematically
at each grid point. However, this method involves large
sampling time due to its inherent exhaustiveness and hence
we opted for low precision (or standard precision) docking in
this step. Based on the results from low precision blind docking
Molecular docking studies
Understanding the binding mechanisms of amyloid modulators
has been of interest for the rational design of small molecule
inhibitors of Aβ aggregation.10,12,16,29 There are reports that
show ridges and grooves on Aβ fibrils serve as binding regions
for ligands, especially the ‘flat’ ligands.30,29,31 From this point of
view, we investigated the mechanisms of groove binding using
our rationally designed NDI conjugates with various functional
groups. We show the correlation of various functional groups
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studies, we selected binding hot spots. Further, we performed 5.09 kcal/mol, respectively for the same site, while GNG and
extra precision (XP) docking and induced fit docking (IFD)35,36 ANA did not show any docked poses. For KLVFFA region, NLD
for the selected hot spots to understand the details of possible showed the highest score of -6.67 kcal/mol followed by WNW
binding sites and the docked ligands.
(-5.56 kcal/mol). FNF, ANA and GNG showed -5.17, -4.81, -4.21
Several structures have been proposed for Aβ aggregates kcal/mol glide docking scores, respectively. NDP had an
based on solid-state NMR experiments.37,38 To select the unexpectedly low score of -3.18 kcal/mol for KLVFFA (Fig. 3C).
receptors for docking studies, the Aβ aggregate structures With the above results in hand, we further performed IFD
available in Protein Data Bank (PDB) were analysed in terms of analysis35,36 wherein receptor was also made flexible in nature.
experimental conditions and length of the peptide used.
Table 1. Qualitative comparison of docking scores for ligands with respect to the
Although PDB ID 2BEG38 (Fig. S5) matched these two criteria,
functional groups
the structure is refined from Leu17 onwards and has only 5
refined chains which represent an insufficient length of binding
groove for our ligands. However, there are other solid-state
NMR structures of Aβ40 in PDB with 6 refined chains stacked
over each other which give sufficiently long grooves on the
surface. We verified and confirmed that the side chain
orientations of these Aβ40 structures match with those
observed in 2BEG (which is Aβ42). PDB IDs 2LMN, 2LMO are
two such Aβ fibril structures which have six refined chains (Fig.
S4) and we have chosen 2LMO for our docking studies.
Nevertheless, we performed docking with 2BEG and obtained
similar docked poses but with lower docking scores which can
be attributed to the missing hydrogen bonds as compared to
the ligands docked to 2LMO. This result revealed that longer
grooves (or binding sites) are necessary to obtained correctly
docked poses of our NDI conjugates. Residue-wise Cα RMSD
(Root Mean Square Deviation) analysis for the NMR structures
The results were found to be similar compared to XP docking
showed two regions viz., residues 13-22 and residues 29-38,
and are tabulated in Table S3 and S4. Preference of various
with distinctly low RMSD values as compared to other regions
NDI conjugates (as obtained from IFD) towards the two
in the selected receptor model sequences (Fig. S5). These two
binding sites is compared in Fig. 3D.
regions are the β-strand forming regions in the Aβ sequence
NDI conjugates-receptor interaction analysis
and have been consistently shown to form β-sheets in many
fibril structures.37-40 The low RMSD regions also include two
NDI conjugates-receptor interaction analysis was performed to
understand the effect of various functional groups on docking
sequence stretches, viz. Aβ16-20 (KLVFFA) and Aβ31-35
preferences of the NDI conjugates. The docking results showed
(IIGLM) that promote the formation of β-sheets in amyloid
fibrils.40
different binding mechanisms adopted by various NDI
conjugates based on the functional groups attached to NDI
Analysis of docked poses in the two low RMSD regions
core. The correlation between the docking scores and the
showed preferential binding of our NDI conjugates with
functional groups suggests a modular and combinatorial
varying degrees of docking scores and selectivity. Results of
method for designing Aβ inhibitors (Fig. 3B). The carboxylic
blind docking studies showed a correlation between the
groups of NDI modulators appeared to be important for
docked poses of the NDI conjugates and two key regions of Aβ
binding of NDI conjugates to KLVFFA region suggesting their
fibril model, namely KLVFFA and IIGLM. All NDI conjugates
role in forming hydrogen bonds and salt bridges, specifically
could be docked to KLVFFA region, whereas, IIGLM region did
with lysine residues (Fig. 3B). NDP which lacks carboxylic
not show any docked poses of ANA and GNG. We have
functionality among all the NDI conjugates showed lowest IFD
extended these results using XP and IFD to investigate the
scores for KLVFFA region. On the other hand, the carboxylic
interactions of NDI conjugates in the two regions. For XP
groups do not seem to be involved in any hydrogen bond or
docking, we used rigid receptor and flexible NDI conjugate
salt bridge interaction in the IIGLM region. However, carboxylic
which showed highest docking score for NLD and NDP
groups might assist NDI conjugate binding by protecting the
followed by WNW and FNF docked to IIGLM site, while there
hydrophobic ligand-receptor interface from being exposed to
was a difference in the scores for KLVFFA site with NLD
the solvent. The influence of hydroxyl groups attached to the
showing highest score followed by FNF, WNW, ANA and GNG
phenyl rings is more pronounced in NDI conjugates docked in
IIGLM region. In general, the hydrogen bond donating
potential on the aromatic rings of the side chains appears to
make hydrogen bonds with Aβ backbone. FNF showed
considerably low docking scores in IIGLM region compared to
NLD, NDP and WNW which have hydrogen bond donors on the
(Fig. 3. and Table S1 and S2). Interestingly, NDP showed the
least preference for binding interaction towards KLVFFA.
Overall, XP docking results showed highest docking preference
for NLD followed by NDP with a glide docking score of -8.37
kcal/mol and -6.95 kcal/mol, respectively for IIGLM site. WNW
and FNF showed glide docking scores of -5.29 kcal/mol and -
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aromatic rings in the imide functional groups. In KLVFFA region control were studied at varying concentrations (20-200 μM).
the hydrogen bond donors of NLD, NDP and WNW form side The results from this study showed potent free radical
chain hydrogen bonding interactions, however their scavenging activity of NLD and NDP (>80% and ~70%
contribution to docking scores appears to be masked by the
respectively), while that of WNW and ANA was in the range of
dominating effect of carboxylic groups. In general, the 20-25% (Fig. 4).
hydrogen bond donors on the aromatic rings of the side chains
Ascorbic acid (0 to 250 µM) showed highest antioxidant
of the imide functional groups seems to make hydrogen bonds property with free radical scavenging potential of ~85% at the
with Aβ backbone. The aromatic ring systems are also initial reading. At the same time point, activities of NLD and
important for better docking as it was inferred from the poor NDP (0 to 250 µM) were 20-30% (Fig. 4). The positive control
docking scores for both ANA and GNG in KLVFFA region and no
docking poses in IIGLM region. The results are qualitatively
summarised in Table 1 which shows the correlation of docking
preferences of individual NDI conjugate. A closer look at Table
1
shows that NDI conjugates with aromatic ring and hydrogen
bond donating groups gave best docking scores (NLD, WNW
,
NDP) with maximum non-covalent interactions in KLVFFA and
IIGLM regions of Aβ fibrils. Interestingly, these in silico
observations are in agreement with the experimental results
which showed superior aggregation-modulation activity for
Fig.
4 DPPH free-radical scavenging (antioxidant) assay for NLD, NDP, WNW, ANA
using Ascorbic acid as standard 0 h (A) and 2 h (B). Values shown are means ±
SEM of three independent experiments performed in three to four replicates.
Radical scavenging activity was expressed as inhibition percentage and was
calculated using the formula: % scavenging activity = (absorbance of control −
absorbance of sample)/ (absorbance of control)] × 100.
NLD, WNW and NDP. From in silico and in vitro results, it can
be generalised that compounds that have three main
characteristics, viz., aromatic rings, hydrogen bond donating
potential on the aromatic rings and the carboxylic groups, are
likely to be the best Aβ42 aggregation modulators. NDI
conjugates which showed poor docking scores for IIGLM and
KLVFFA were found to be experimentally inactive in both
inhibition and disaggregation of Aβ aggregates. It is evident
from the above results that various functional groups attached
to the NDI core of individual compounds show different
preferences based on their interaction potentials with the
selected regions of aggregates. Although computational
docking has certain limitations in accurately predicting the
binding sites/regions, the results from both docking and in
vitro experiments have converged to similar findings. Taken
together, current investigation of a series of NDI conjugates
with varying functionalities provided significant information on
the characteristics of aggregation modulators and paves a new
path for the design of novel NDI modulators through a
combinatorial approach.
ascorbic acid continued to show good radical scavenging
activity (~80%) at 2 h and the activities of NLD and NDP were
comparable at this point. Interestingly, NLD and NDP showed
higher antioxidant property (~80%) at lower concentrations (<
50 μM) while ascorbic acid exhibited ~35% activity at similar
concentration (Fig. 4). These results suggested that NLD and
NDP show steady and sustained antioxidant activity compared
to ascorbic acid at lower concentrations. On the other hand,
the observed low antioxidant property of WNW and ANA is
due to absence of phenolic hydroxyl groups. Overall, NLD and
NDP showed good antioxidant activity which may play key role
in reducing the oxidative burden caused by ROS and prevent
the progression of multifaceted Aβ aggregation process.
Aβ42 aggregates induced toxicity in neuronal cells
After successfully establishing the modulation effects of NDI
conjugates, we next sought to evaluate the effect of lead
modulators NLD and NDP in cellulo conditions. First, the
cytotoxic effects of pre-formed Aβ42 aggregates was assessed
in pheochromocytoma cells (PC12) of the rat adrenal medulla.
PC12 cell lines are neuroblastic cells and a good model for
neuronal cells.43 In this experiment, Aβ42 (100 μM) in PBS (10
Antioxidant property of NDI modulators
Elevated levels of ROS aggravates Aβ aggregation and vice
versa which results in biomolecule damage and neuronal cell
death.41 Development of Aβ modulators supplemented with
antioxidant properties is one of the widely explored strategies
towards AD treatment.16,17 It is well-known that polyphenols
and flavonoids based natural compounds show good
mM, pH 7.4) was incubated for 24 h at 37 °C to obtain Aβ42
fibrillar aggregates. PC12 cells were treated with preformed
Aβ42 aggregates at variable concentrations (10, 7.5, 5, 2.5 and
1 μM) for 24 h (Fig. S6). The main objective of this experiment
was to determine the optimal concentration of Aβ42
aggregates that cause ~50% cell death. The cellviability of PC12
cells after Aβ42 treatment was accessed through MTT assay.
Concentration dependent toxicity was observed in the PC12
cells with 1-10 μM of Aβ42 aggregates exhibiting ~ 55-70%
cytotoxic effect. Based on this study, 1 μM of Aβ42 exhibiting
45% cell viability (55% cytotoxicity) was considered optimum
for further experiments.
antioxidant property.42 The NDI modulators with L-dopa (NLD
and dopamine NDP closely resemble polyphenolic
)
(
)
compounds in terms of their structure (hydroxyl groups on the
aromatic ring), function as aggregation modulators and
possibly provide antioxidant effect. Keeping this in mind, NLD,
NDP
in DPPH free radical scavenging assay. In this experiment, NLD,
NDP WNW and ANA along with ascorbic acid as a positive
, WNW and ANA were assessed for antioxidant property
,
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NLD and NDP rescue neuronal cells from Aβ42 toxicity
PC12 cells were treated with Aβ42 (1 µM):NLD/NDP (1 µM) for
24 h at 37 ⁰C (Fig. 5B). The cells treated with Aβ42 aggregates
showed ~50% cell death, while the cells co-incubated with
Aβ42 aggregates and NLD showed only 35% cell death, a
significant reduction in cell death by ~15% (Fig. 5B). NDP also
showed a significant reduction in cell death by ~10% and slight
reduction in the rescue activity could be attributed to the
absence of carboxylic group compared to NLD. Thus, NLD is
the most potent NDI modulator for rescuing neuronal cells
from Aβ42 toxicity through both aggregation inhibition and
dissolution of pre-formed Aβ42 aggregates.
We assessed the ability of NLD and NDP to ameliorate the
cytotoxicity induced by Aβ42 aggregates in PC12 cells. PC12
cells treated with aggregates prepared from
1 μM
concentration of Aβ42 showed ~55% cytotoxicity similar to the
toxicity profile obtained in concentration-dependent study
(Fig. 5A). The cells co-incubated with NLD (1 μM) and Aβ42 (1
Conclusions
In conclusion, we have developed natural amino acids, L-dopa
and dopamine conjugated NDIs with dual functionality viz.,
modulation of Aβ42 aggregation and antioxidant property. ThT
fluorescence assay and dot blot analysis revealed that NLD
with L-dopa is an efficient modulator of Aβ42 aggregation and
rescue neuronal cells from Aβ42 toxicity. In silico molecular
docking results are found to be consistent with the in vitro and
in cellulo studies. The molecular docking study showed the
presence of hydrogen bonding and hydrophobic interactions of
NLD in the hydrophobic pockets of Aβ42 aggregates, which is
attributed to its efficient modulation of Aβ42 aggregation.
Furthermore, the excellent antioxidant property of NLD as
Fig. 5. In vitro studies with Aβ42 and NDI modulators NLD and NDP. (A) The
effect of NDI modulators, NLD and NDP against Aβ42 aggregates on the cell
viabilities of PC12 cells. Aβ42 either alone or with NLD, NDP are incubated for 24
h at 100 μM concentration at 1:1 ratio in PBS. PC12 cells were treated with 1μM
(final concentration per well w.r.t either Aβ or drug) above samples for 24 h and
assayed by MTT. (B) The effect of NDI modulators, NLD and NDP in
disaggregation of pre-formed Aβ42 aggregates on the cell viabilities of PC12 cells.
200 μM of Aβ42 alone in PBS was incubated for 24h followed by co-incubation
with NLD, NDP for another 48 h at 100 μM concentration at 1:1 ratio in PBS.
PC12 cells were treated with 1 μM (final concentration per well w.r.t either Aβ or
drug) above samples for 24 h and assayed by MTT. One-way ANOVA analysis studied by the antioxidant scavenging assay, significantly
followed by Tukey’s multiple comparison post hoc test was performed (**p <
contribute towards the rescue of neuronal cells from Aβ42
0.001 compared to control in a, b & c; **p < 0.001, *p < 0.01 and #p < 0.05
induced oxidative stress. Overall, the strategic design and easy
compared to Aβ42 aggregates group in A & B).
access to NDI conjugates through simple synthetic routes and
their activity-evaluation through various in vitro, in silico and in
μM) aggregates showed a significant reduction in cell death
cellulo studies revealed NLD as a potent modulator of
(~30%), while NDP showed ~20% reduction (Figure 5A). The
multifaceted Aβ42 toxicity.
improved cell viability exhibited by NLD and NDP can be
attributed to their anti-aggregation and antioxidant (phenolic
groups) properties. Notably, NLD showed relatively superior
Experimental section
cell viability against toxicity induced by Aβ42 aggregates
Materials
compared to NDP. At this point, mechanism of action of NLD
and NDP responsible for the observed inhibitory activities was PC12 Adh cells (a rat adrenal pheochromocytoma cell line)
not fully understood. The only structural difference between were kind gift from Dr. Praveen Vemula laboratory, inSTEM,
NLD and NDP is the presence of carboxylic acid functionality in NCBS, Bengaluru. Roswell Park Memorial Institute (RPMI) 1640
the former which seems to play key role in the improved cell media, heat-inactivated horse serum, fetal bovine serum,
viability. We attempted to explain the observed modulation penicillin-streptomycin and trypsin were purchased from Gibco
data and the possible role of carboxylic acid functionality of (Grand Island, NY, USA). Aβ42 peptide was purchased from
NLD in enhancing the activity, by performing docking studies. Calbiochem, Merck. Rink amide resin, Fmoc-amino acids were
The carboxylic group of NDI modulators play a vital role in purchased from Novabiochem, USA. Dimethyl sulfoxide,
interaction with KLVFFA region through hydrogen bonding and methyl thiazolyl blue tetrazolium bromide (MTT), Dulbecco’s
salt bridge formation with the lysine residues of KLVFFA phosphate-buffered saline (PBS) were purchased from Sigma-
region, thus improving binding affinity. NDP lack carboxylic Aldrich. Dimethyl formamide and isopropanol were purchased
group, which has hampered its strong binding towards KLVFFA from Acros Organics. DIPEA, HBTU, piperidine were purchased
region leading to lower aggregation modulation efficiency and from Spectrochem, India. All the other chemicals and reagents
cell viability over NLD
.
were used as received unless otherwise mentioned.
Next, we assessed the ability of NLD and NDP to modulate General procedure for the synthesis of amino acid and dopamine
the pre-formed toxic aggregates in dissolution (disaggregation) conjugated NDIs (GNG, YNY, NLD and NDP)
experiments (Fig. S7). First, Aβ42 (200 μM) was subjected to
1,4,5,8- Naphthalenetetracarboxylic dianhydride (NDA) (0.5 g,
aggregation for 24 h followed by co-incubation with NLD and
1.8 mmol) and corresponding amino acid/dopamine (3.7
NDP at 100 μM concentration (at 1:0.5 ratio) for 48 h. The
mmol) were suspended in 10 mL of dimethylformamide (DMF).
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To this suspension, triethylamine (TEA) (0.2 mL) was added and further dried under vacuum. HFIP-treated Aβ11-25 or
and allowed to reflux for 12 h. After cooling to room Aβ42 was then dissolved in PBS buffer to a final concentration
temperature and DMF was removed under high vacuum. The of 100 μM at pH 7.4 and vortexed for approximately 30 s. NDI
reaction mixture was added with 500 mL of 2N hydrochloric conjugates were dissolved in PBS buffer to
a final
acid (HCl) and was stirred for 1 h. The precipitate was collected concentration of either 200 μM or 100 μM. For inhibition
through buchner funnel attached with suction, washed with studies involving Aβ11-25, 100 μM peptide was either added
excess of distilled water (to remove excess HCl and other to the NDI conjugates at 1:1 ratio or incubated alone for 48 h
starting materials) and dried under vacuo to obtain amino at 37 ⁰C. For studies involving inhibition of Aβ42 aggregation,
acid-naphthalenediimide (NDI) conjugates (GNG, YNY, NLD) in Aβ42 (50 μM) was either added to NDI conjugates at 1:1 ratio
quantitative yield. For NDP, acidification step with HCl was or incubated alone for 24 h at 37 ⁰C. Samples from each
excluded. The reaction mixture after DMF removal was added experiment were analysed by standard ThT fluorescence assay.
with water and directly filtered using buchner funnel and Aggregates dissolution studies
precipitate was washed with water several times and the
Aβ11-25 or Aβ42 peptide in PBS were dissolved to a final
product was dried to obtain NDP in quantitative yield.
concentration of 200 or 100 μM and incubated for 24 or 48 h
Synthesis of ANA, FNF and WNW was reported in our earlier
publication.44 All the products were thoroughly characterised
at 37 ⁰C for the formation of aggregates/fibrils respectively for
each peptide. For dissolution studies involving Aβ11-25, the
by NMR and mass spectrometry.
peptide at 200 μM was incubated for 48 h followed by co-
GNG: Yield: 70%; 1H NMR (DMSO-d6, 400 MHz) δH 13.20 (2H,
incubation with NDI conjugates for another 24 h or for 6 days
b), 8.74 (4H, s), 4.77 (4H, s).; 13C NMR (DMSO-d6, 100 MHz)
(for time and dose dependent study) at 100 μM final
δC 168.9, 162.2, 130.9, 126.2, 126, 41.5; HRMS: found
concentrations (1:1 ratio). For control, 100 μM Aβ11-25 was
382.0437 [M+H]+, calcd. 382.0437 for [C18H10N2O8+ H].
incubated alone for 72 h. Dissolution studies with Aβ42, 100
YNY: Yield: 75%; 1H NMR (DMSO-d6, 400 MHz) δH 12.99 (2H,
μM of peptide was initially incubated for 24 h followed by co-
b), 9.04 (2H, s), 8.65 (4H, s), 6.94 (J=1.15, 4H, d), 6.49 (J=1.72,
incubation with NDI conjugates for another 48 h at 50 μM final
4H, t), 5.78 (J=2.55, 2H, q), 3.48 (2H, m), 3.24 (2H, m); 13C
concentration (1:1 ratio). For control, 50 μM Aβ11-25 was
NMR (DMSO-d6, 100 MHz) δC 170.2, 161.8, 155.5, 131.1,
incubated alone for 72 h. Disaggregation of pre-formed fibrils
129.7, 127.6, 125.9, 125.5, 114.9, 54.7, 33.2; HRMS: found
in the presence of NDI conjugates was studied by ThT
595.1397 [M+H]+, calcd. 594.1274 for [C32H22N2O10+H+].
fluorescence assay.
NLD: Yield: 75%; 1H NMR (DMSO-d6, 400 MHz) δH 12.98 (2H, s),
ThT assay (Fluorescence spectroscopy)
8.66 (4H, s), 8.61 (2H, br), 8.52 (2H, br), 6.53-6.35 (8H, m), 5.74
(2H, q), 3.38 (2H, dd), 3.16 (2H, dd); 13C NMR (DMSO-d6, 100
Fluorescence spectral measurements were carried out using
Perkin Elmer Model LS 55 fluorescence spectrophotometer.
MHz) δC 170.3, 161.9, 144.7, 143.5, 131.2, 128.4, 126.0, 125.6,
119.6, 116.2, 115.3, 54.8, 33.5; HRMS: found 627.1248 [M+H]+,
Maximum fluorescence of ThT was observed with the
excitation and emission wavelengths set to 450 and 482 nm,
calcd. 627.1246 for [C32H22N2O12+ H].
NDP: Yield: 75%; 1H NMR (DMSO-d6, 400 MHz) δH 8.81 (2H, s),
respectively. A ThT concentration of 5-10 μM was used for
Aβ42 fibrillation and dissolution assays based on the fibrillar
8.67 (2H, s), 8.65 (4H, s), 6.68-6.62 (4H, m), 6.50 (2H, q), 4.17
(2H, dd), 2.75 (2H, dd); 13C NMR (DMSO-d6, 100 MHz) δC 162.3,
concentration.
Transmission Electron Microscopy (TEM)
145.1, 143.7, 130.3, 129.2, 126.1, 125.9, 119.2, 115.9, 115.6,
41.7, 32.7; HRMS: found 539.1451 [M+H]+, calcd. 539.1449 for The samples of Aβ42 aggregates or Aβ42-modulator (NDP or
[C30H22N2O8+H].
NLD) were diluted to 5 µM in milliq water. Then 5 μL of each
sample was adsorbed onto 200-mesh TEM grids for 10 min and
washed for 1 min with distilled water. The samples were
stained with uranyl acetate (2%) for 5 min and washed for 3
times with distilled water. The samples were dried overnight
and visualised under TEM operating at 120 kV.
Cell culture and treatments
PC12 Cells were cultured in T-75 flasks (Nest, India) and
maintained in RPMI 1640 medium supplemented with 10%
heat-inactivated horse serum, 5% fetal bovine serum, 100
U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in 95%
air/ 5% CO2. Stock solutions of NLD (10 mM) and NDP (10 mM)
were prepared in dimethylsulfoxide (DMSO) and aliquots were
Molecular docking
stored at -20 °C. For all experiments assessing protective The Glide program in Schrödinger Software Suite was used for
effects of NLD and NDP, PC12 cells were pre-treated with all the docking studies. The general pipeline for docking used
indicated concentrations of compounds for 24 h and then in this work is as shown in ESI. In order to perform an unbiased
assayed for MTT unless stated otherwise. Controls were search for possible binding sites for NDI conjugates on the Aβ
treated with media containing 0.01% DMSO.
surface, we adopted computational blind docking strategy
followed by extra-precision and induced fit docking for
selected sites (see ESI for detailed blind docking protocol).
Since the ligands in this study were new ligands, it was
necessary to confirm the favourable binding regions using
blind docking. In contrast to usual docking studies where the
Aggregation inhibition studies
Aβ11-25 (see ESI for synthetic details) or Aβ42 peptide (0.25
mg) (Calbiochem, Merck, USA) were dissolved in hexafluoro-2-
propanol (HFIP, 0.2 mL) were incubated at room temperature
for 1 h. HFIP was then removed by the thin flow of nitrogen
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binding site is known, blind docking is an iterative method absorbance was measured at 570 and 690 nm using microplate
designed to scan the whole surface of the receptor uniformly. reader (Spectramax i3x, Molecular device). Background-
This procedure was automated through in-house python corrected values (570−690 nm) were used to plot the graph.
scripts that generate an equispaced grid over the surface of Data from at least three experiments were analysed using
the receptor and test the docking performance at each grid GraphPad software.
point systematically. However, this method involves large Modulation of Aβ42 toxicity in PC12 cells
sampling due to its inherent exhaustiveness. Hence, low
Aβ42 alone (10 μM) was able to induce around
∼60% cell
precision (or standard precision) docking (available in Glide
program provided in Schrödinger Software Suite) was
performed in this step. Based on the results from low precision
blind docking studies, binding hot spots were selected. Extra
precision (XP) docking and induced fit docking (IFD) were
performed subsequently for the selected hot spots to
understand the details of the docked poses of the ligands.
Protocol for blind docking is given in the supplementary
information.
death after incubating for 24 h. Therefore, we assessed the
ability of active NDI modulators, NLD and NDP to ameliorate
the cytotoxicity induced by Aβ42 (10 μM) after incubating for
24 h. The ability of NDI modulators to disaggregate the pre-
formed fibrils was evaluated by the treatment of PC12 cells
with NLD and NDP, for protective effects. For these
experiments, Aβ42 was incubated with/without NDI modulator
either in monomeric form (aggregation inhibition) or pre-
formed aggregates (dissolution of aggregates).
For
DPPH radical scavenging assay (Antioxidant activity)
aggregation inhibition experiment, 50 μL of Aβ42 (100 μM)
To a 96-well plate, 100 μL aliquot of solution of NDI modulator was mixed with 50 μL of NDI modulator (100 μM) to yield 50
(dissolved in methanol) ranging from 250 to 20 μM was added. μM final concentration, vortexed for approximately 30 s and
Next 100 μL of 200 μM methanolic solution of 1,1-DPPH was incubated for 24 h at 37 ⁰C. For dissolution experiment, pre-
added, and the plate was shaken at 30 ⁰C for 30 min. Control formed Aβ42 aggregates from 24 h incubation were further
wells received 100 μL of methanol and 100 μL of 200 μM incubated either alone or with 100 μM of NDI modulator for
methanolic DPPH solution. Wells containing only 200 μL of another 48 h. These samples were treated to PC12 cells at 1
methanol served as a background correction. The change in μM final concentration (Aβ42: NDI modulator) in DMEM
absorbance of all samples and standard (ascorbic acid) was complete media for 24 h. After 24 h of incubation, MTT assay
measured at 517 nm. Absorbance was measured at every 30 was performed as mentioned above to assess the protective
min. For 0 min, absorbance was measured immediately after effects of NDI modulators.
addition of DPPH solution. Radical scavenging activity was Statistical analysis
expressed as inhibition percentage and was calculated using
Experimental data presented is the mean ± SD of at least three
the following formula:
% scavenging activity = (absorbance of control −absorbance of
independent experiments performed with 4–5 replicates. One-
way ANOVA analysis followed by Tukey’s multiple comparison
sample)/ (absorbance of control)] × 100.
post hoc test was performed. Data were compared among the
Cytotoxicity of Aβ42 aggregates in PC12 cells
different groups for individual experiment and p < 0.05 was
Aβ42 peptide was dissolved in PBS buffer to
a final considered as significant.
concentration of 50 μM and incubated at 37 ⁰C for 24 h. The
samples from 24 h incubation were used to evaluate the effect
of various Aβ42 aggregation species on the viability of PC12
cells (extracellular toxicity). Briefly, PC12 cells were seeded at
17,000 cells/well density in 100 μL of the medium in a 96-well
plate. Cells were allowed to adhere to the plate for 24 h. 24 μL
(100 μM, Aβ42 ) sample was diluted with PC12 cell medium to
prepare variable concentrations of Aβ42 (1, 2.5, 5, 7.5, and 10
μM) for cell culture experiments. Media was removed, and the
adhered PC12 cells were treated with 60 μL of Aβ42 (1-10 μM)
containing medium. For evaluating the effect of Aβ42 + NDI
modulator, samples were diluted with PC12 medium to get the
final concentration of Aβ42 and compound to 10 μM in PC12
medium. Control cells were treated with appropriately diluted
PC12 medium with PBS. Treatment with extracellular Aβ42 was
conducted for 24 h. After incubation, 6 μL of 5 mg/mL MTT
was added to the cells and the plate was incubated for another
4 h at 37 ⁰C under 5% CO2 atmosphere. Next, the plate was
centrifuged at 1500 rpm for 10 min and the supernatants were
carefully removed. The formazan crystals were dissolved in
100 μL of 1:1 mixture of DMSO/methanol solution by shaking
gently at 400 rpm for 30 min at room temperature. Then the
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank Prof. C. N. R. Rao FRS for constant support and
encouragement, JNCASR, SwarnaJayanti Fellowship, the
Department of Science and Technology (DST), Government of
India (Grant: DST/SJF/CSA-02/2015-2016) and Sheikh Saqr
Laboratory (SSL), ICMS-JNCASR, for financial support.
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Graphical Abstract
L-Dopa
and
dopamine
napthalenediimides modulate amyloid
conjugated
toxicity
β
Madhu Ramesh, Pandeeswar Makam, Chandrashekhar
Voshavar, Harshavardhan Khare, Kolla Rajasekhar,
Suryanarayanarao Ramakumar, Thimmaiah
Govindaraju*
We report amino acids, L-dopa and dopamine
functionalised naphthalenediimides and the detailed in
silico and in vitro study to identify potential
multifunctional modulator of amyloid
β
toxicity.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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Organic & Biomolecular Chemistry
Page 12 of 12
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DOI: 10.1039/C8OB01691G
ARTICLE
Journal Name
12 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
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