Diphenyl triazine hybrids inhibit α-synuclein fibrillogenesis: Design, synthesis and in vitro efficacy studies

Article abstract of DOI:10.1016/j.ejmech.2020.112705

Aggregation of α-synuclein (α-syn) is one of the central hypotheses for Parkinson's disease (PD), therefore, its inhibition and disaggregation is an optimistic approach for the treatment of PD. Here, we report design, synthesis and in-vitro efficacy studi

Full text of DOI:10.1016/j.ejmech.2020.112705

European Journal of Medicinal Chemistry 207 (2020) 112705
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Diphenyl triazine hybrids inhibit
a-synuclein fibrillogenesis: Design,
synthesis and in vitro efficacy studies
a
b
b
b, **
Mudasir Maqbool , Joshna Gadhavi , Pravin Hivare , Sharad Gupta
Nasimul Hoda
a
,
a, *
Drug Design and Synthesis Lab., Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110025, India
Biological Engineering, Indian Institute of Technology Gandhinagar, Palaj, Gandhinagar, Gujarat, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 May 2020
Received in revised form
Aggregation of a-synuclein (a-syn) is one of the central hypotheses for Parkinson’s disease (PD),
therefore, its inhibition and disaggregation is an optimistic approach for the treatment of PD. Here, we
report design, synthesis and in-vitro efficacy studies of a series of diphenyl triazine hybrids as potential
13 July 2020
inhibitors of
a-syn fibrillogenesis. From the docking studies, we concluded that compounds A1, A2, A4,
Accepted 28 July 2020
Available online 22 August 2020
A8 and A9 display promising binding affinity with the essential residues of
a-syn with binding energy
values: ꢀ6.0, ꢀ7.0, ꢀ6.3, ꢀ6.6 and ꢀ6.7 kcal/mol respectively. The target compounds were synthesized
using multistep organic synthesis reactions. Compounds A1, A2 A4, A8 and A9 showed a significant
Keywords:
lowering of the
these compounds A1, A2, A4, A8 and A9 also proved to be good disaggregators in the pre-aggregated
form of -syn. Most of the compounds exhibited no cytotoxicity in mouse embryonic fibroblast (MEF)
a-syn fibril formation during Thioflavin-T assay and fluorescence microscopy. In addition,
Neurodegeneration
Parkinson’s disease
Protein aggregation
Drug design
a
and human adenocarcinomic alveolar basal epithelial cells (A549) except A2. Overall, diphenyl triazine-
Synthesis
Diphenyltriazine
based compounds can be further investigated for the treatment of synucleinopathies and for Lewy body
dementia in which a-syn is predominantly observed.
©
2020 Elsevier Masson SAS. All rights reserved.
1. Introduction
Presence of some of these conformers has been confirmed in the
diseased brain tissues as well [7]. Therefore, robust inhibitors that
Parkinson’s disease (PD) is the second most common neurode-
could not only avert the fibrillization of
a
-syn but also disaggregate
generative disorder after Alzheimer’s Disease (AD) which primarily
occurs due to the loss of dopaminergic neurons in the substantia
nigra region of the brain [1]. The two major pathological hallmarks
formerly accumulated aggregates of
continue to be an active field of investigation. Researchers have
reported different synthetic and natural products that can
a-syn in the human brain
of PD are Lewy bodies and Lewy neurites in which
syn) protein has been found to be the major constituent [2].
40 amino acid residues long which is a natively unfolded in so-
lution and plays a pivotal role in vesicle trafficking [3,4] Aggrega-
tion of -syn is one of the central events in the onset and
progression of PD and hence in-vitro aggregation is extensively
used as a model system to study PD [5]. -Syn aggregation is a
multi-step process following nucleation dependent aggregation
kinetics that undergoes various stages of aggregation such as
nuclei, oligomers, proto-fibrils, fibrils and larger aggregates [6].
a
-synuclein (
a
-
constrain a-syn aggregation and ameliorate the aggregation asso-
ciated toxicity [8e10].
a-Syn is
1
1,2,4-triazine based compounds were found to be proficient in
penetrating BBB and advantageous agents for preventing neuro-
degenerative diseases like AD [11,12]. Such compounds were also
found to be neuroprotective [13]. In our efforts towards the
development of potent multitarget ligands for the treatment of AD,
a series of triazine-triazolopyrimidine hybrids was reported that
effectively inhibited Ab42 peptide aggregation by 1e1.4 folds better
as compared to the effect of curcumin [14]. One more series of
cyanopyridine-triazine hybrids also showed better inhibitory ac-
a
a
tivity for Ab42 aggregation (1.09e1.5 folds) at 25 mM compared to
the reference compound curcumin [15]. The plausible reason for
their remarkable anti-aggregation activity may be due to the
presence of core triazine moiety through which the
*
Corresponding author.
** Corresponding author.
E-mail addresses: sharad@iitgn.ac.in (S. Gupta), nhoda@jmi.ac.in (N. Hoda).
https://doi.org/10.1016/j.ejmech.2020.112705
223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
0

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
pharmacophore acquires a nearly planer structure which in turn
enables it to fit between the Ab sheets and thus produced an Ab
disaggregating effect [15]. Hydrazinyl-1,2,4-triazines bearing
pendant aryl phenoxy methyl-1,2,3-triazole were correspondingly
alkyl or aralkyl groups with varying hydrophobicity and electronic
properties also dictated the binding affinity of the ligands to the
drug target. The binding free energy of the top five compounds A1,
A2, A4, A8 and A9 with
a
-syn was found to be ꢀ6.9, ꢀ7.2, ꢀ6.6, ꢀ7.0
found to inhibit BASE1 (
b-secretase) efficiently. Furthermore, these
and ꢀ6.7 kcal/mol, respectively which suggest a very high binding
affinity between the ligands and the target protein. PyMOL viewer
was used for the analysis of the docked structures. From the
interpretation of the docking results as shown in Fig. 2A, compound
compounds were found to be neuroprotective against A
b
peptide
toxicity [16]. Recently, the multifunctionality of 5,6-diphenyl
triazine-thio methyl triazole hybrids for the anti-Alzheimer effect
was also evaluated [17]. The easy synthesis of triazine-based
compounds by routine chemical reactions and the roughly
planner structure of triazine core structure were also the reasons to
A1 was found to participate in
p-p interaction between chlor-
oquinoline system and Tyr136. Same way, one of the biphenyl rings
attached to the triazine core structure was found to interact with
Tyr125. From Fig. 2B, we came to know that, biphenyl and triazolo-
choose such compounds as a-syn disaggregating agents.
The rationale for designing diphenyltriazine-based compounds
for the anti-PD effect is diagrammatically illustrated in Fig. 1.
Keeping the above aspects into consideration, here we have
designed and synthesized a series of diphenyltriazine hybrids as
pyrimidine systems of A2 displayed
Tyr136, the key residues of the protein, respectively. The biphenyl
rings attached to the triazine were found to show interaction
with Tyr125, simultaneously (Fig. 2C). In the same manner, A8
displayed a non-covalent interaction with Tyr125. Also, H-bonding
interaction of the range of 1.8 Å was observed between the nitrogen
p-p stacking with Tyr125 and
p-p
persuasive inhibitors of
for the treatment of PD. The in-vitro ability of the synthesized
compounds to inhibit -syn fibrillogenesis was evaluated. The
a-syn fibrillation and disaggregating agents
a
of phenylacetamide of A8 and Met127 (Fig. 2D). In addition to p-p
compounds were also tested for their cytotoxicity effects on MEF
cell lines and A549 cell lines.
interaction between the aromatic groups of A9 and Tyr125 and
Tyr136, a strong H-bond was detected between the oxygen of
phenylacetamide and hydrogen of Ser129 (Fig. 2E).
Bolded values represent the most active binding compounds:
A1, A2, A4, A8 and A9.
2
. Results and discussion
2.1. In silico design of diphenyl-triazine based a-syn inhibitors
2.2. Chemical synthesis
To develop a potential drug candidate for PD which could inhibit
-syn aggregation, we undertook the structure-based drug design
a
The target molecules were synthesized via multiple steps as
approach. AutoDock Vina version 1.5.6 was used for flexible dock-
ing and redocking of the diphenyl triazine-based molecules and the
micelle-bound human a-syn (PDB ID 1XQ8).a-Syn is a 140 amino
acid long, small and natively unfolded protein which can be divided
into three sequential parts: N-terminal amphipathic region con-
taining maximum sequences and three point mutations associated
with autosomal dominant early onset PD, the central NAC region
which covers the most hydrophobic residues, and the hydrophilic
C-terminal.
represented in Scheme 1. In the first step benzil (1) was made to
couple with thiosemicarbazide (hydrazinecarbothioamide) (2) in
water and ethanol in equal proportions to get 3 according to the
reported literature [27]. The compound 3 was treated with 2-
chloroacetic acid to get compound 4. Peptide coupling of com-
pound 4 with different piperazine containing nucleophiles in
presence of DMSO as a reaction solvent, triethylamine as base and
HBTU as coupling reagent was the final step of the reaction to
obtain the target molecules (A1eA9). The column chromatography
technique was used for the purification of all the molecules and the
Autoinhibition of a-syn by the interaction of C-terminus region
1
13
with the NAC region has been found to prevent fibrillation under
normal conditions [18,19]. The C-terminal region of the protein has
an essential part in the interaction of protofibrils required for
purified compounds were characterized by H NMR, C NMR, ESI
MS and elemental analysis.
maturation of fibrils [20]. The amino acid residues of
Tyr125, Tyr133, and Tyr136 present in the hydrophilic tail of the C-
terminus of -syn play a crucial role in its fibrilization process
21,22]. Previously, the binding of ceftriaxone to bovine serum al-
a
-syn like
2.3. In-vitro evaluation of the designed and synthesized compounds
(A1eA9) as a-syn aggregation inhibitors
a
[
2.3.1. Expression and purification of a-Syn protein and its
bumin (BSA) has been reported to increase the polarity and hy-
drophilicity of the C-terminal of the protein, thereby, increasing the
characterization
Recombinant tag-free
a-syn was overexpressed in Escherichia
compactness of monomeric
18,23]. Consequently, binding of a putative ligand to the C-termi-
nal region should affect the aggregation of -syn. Even though the
a
-syn and inhibited its fibril formation
Coli BL21 (DE3), and since the protein is primarily localized in the
periplasm, the osmotic shock method was used to extract the
protein as described previously [28]. Further purification of a-syn
[
a
protein topology reveals it in a micelle bound state, the unfolded
hydrophilic tail (Asp98-Ala140) can be well suited as a docking
target [24].
The choice for selecting diphenyl-triazine core moiety was due
to its extended conjugate system and the excellent reports of
triazine-based molecules as anti-neurodegenerative agents [14,25].
The aromatic systems joined through the combination of flexible
and rigid linkers were expected to promote the non-covalent in-
was carried out on DEAE (Diethyl aminoethyl)-Sepharose, an anion
exchange matrix column, using a gradient elution of NaCl with
concentration changing from 50 mM to 500 mM. As shown in
Fig. 3a, SDS-PAGE analysis of various fractions revealed that the
purified protein was primarily present in 300e500 mM NaCl frac-
tions. All these fractions were pooled and dialyzed against water to
yield a salt-free preparation of
fibrillization of -syn is sensitive to the identity and concentration
of salts present [29]. To further ensure the monomeric nature of a-
a-syn with purity >95% as kinetics of
a
teractions like
essential residues of
p
-
p
stacking interaction and H-bonding with the
-syn. Compounds containing various
a
syn before each assay, the salt free protein was first dissolved in
base and then acidified to a neutral pH. This was followed by a high-
speed centrifugation at 100,000 g to remove any residual aggre-
gates. Above steps were necessary to obtain a consistent and
baseline kinetic aggregation profile for comparing the inhibitory
hydrogen bond donor and acceptor groups appeared favorable for
their binding with the protein. The docking results provided the
approximate binding affinity (-
A1eA9) and the micelle-bound human
membrane imbedded conformation as mentioned in Table 1. The
DG in Kcal/mol) between the ligands
(
a
-syn that mimics a
effect of synthesized compounds. The identity of purified a-syn was
2

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
further confirmed by Western blot using H3C monoclonal antibody
which gave a single band with M.Wt. ~17 kDa, as shown in Fig. 3b.
This was higher than theoretical M.Wt. ~14.4 kDa, due to low
apparent lengthening of the lag phase, all compounds A1-A9
caused a decline in the final plateau fluorescence (Ffinal) to varying
extents. Although Ffinal does not relate absolutely to the amount of
binding of acidic C-terminal region of
previously [28].
a
-syn to SDS as observed
amyloid formation, a relative measure of b-sheet rich structures
formed could be inferred and we relied on the % decline in the Ffinal
to compare the inhibitory potential [31,32]. A significant decrease
would indicate that a particular compound can inhibit the fibrilli-
zation under the given aggregation reaction conditions. As shown
2.3.2. In vitro monitoring of
a-syn fibrillization in the presence of
inhibitory compounds (A1-A9)
Thioflavin T (ThT) is a benzothiazole based dye that exhibits
several-fold enhancements in the fluorescence emission upon
in Fig. 5, A1, A2, A8 and A9 significantly inhibited a-syn fibrilliza-
tion as indicated by more than 50% decline in Ffinal (p<0.01%). These
compounds were also top scorers according to our computational
predictions based on docking studies (Table 1). A3, A4, and A7 also
exhibited significant lowering (P < 0.05) but decline was less than
40% in Ffinal. To rule out any interference due to self-aggregation of
the inhibitory compounds, A1-A9 were separately incubated with
binding to the cross
b-sheet structures, a hallmark of amyloids and
hence is widely used to monitor the in vitro aggregation in real-
time [30]. The progress of
a
-syn aggregation (70
mM) was moni-
ꢁ
tored in the presence of ThT at 37 C by capturing fluorescence
emission at 485 nm (excitation at 445 nm) in a microplate spec-
trofluorometer. A sigmoidal ThT fluorescence curve was obtained
ThT only in the absence of a-Syn for more than 2 weeks but no
change in ThT fluorescence was observed (data not shown).
To gain further insights into the mechanism of inhibition by A1-
A9, we calculated lag-time (tlag), time required to reach halfway
through the elongation phase (t1/2) and apparent elongation rate
constant Kapp for the growth of fibrils from ThT fluorescence
sigmoidal curve [33]. As shown in Table 2, except A2, no significant
change in tlag or t1/2 was observed when compared to uninhibited
fibrillization reaction. This indicates that compounds are probably
inhibiting the elongation phase with no significant impact on
for
a-syn aggregation with a distinct lag phase preceding the
growth phase, followed by a plateau phase mimicking a typical
nucleation dependent fibrillization kinetics. To test the inhibitory
potential of synthesized compounds (A1-A9)
was carried out in PB, pH 7.4 supplemented with the inhibitors
a-syn aggregation
dissolved in DMSO, such that the final concentration of DMSO was
10% (v/v) and a-syn to inhibitor molar ratio was fixed at 1:1. As a
positive control, DMSO sans any inhibitory compounds was added
to the aggregation mixture. As shown in Fig. 4, while there was no
Fig. 1. The rationale for the design of diphenyltriazine analogues for inhibiting a-syn fibrillogenesis.
3

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
Table 1
The calculated binding free energies of the selected derivatives in molecular docking using Auto Dock Vina [26].
Compound name
A1
Compound structure
Binding affinity (kcal/mol)
ꢀ6.9
A2
ꢀ7.2
A3
ꢀ6.1
4

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
Table 1 (continued)
Compound name
A4
Compound structure
Binding affinity (kcal/mol)
¡6.6
A5
ꢀ6.0
A6
ꢀ6.4
(continued on next page)
5

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
Table 1 (continued)
Compound name
A7
Compound structure
Binding affinity (kcal/mol)
ꢀ5.4
A8
¡7.0
A9
¡6.7
nucleation or early aggregation. A2 appeared to be the exception as
initial acceleration leading to an earlier onset of elongation phase
was observed. Ffinal for A1 and A2 was nearly identical which
suggested a similar role of bulkier bicyclic ring system on inhibition
also bind to regions other than non-amyloidogenic which in turn
could reduce the inhibitory potential during the early stages of
aggregation. However, as the aggregation proceeds, the stoichio-
metric ratio favors inhibitors and the further extension of the
protofibrils/fibrils is stalled prematurely resulting in lower Ffinal
than positive control. A logical step would have been to use a higher
concentration of the inhibitors but we were limited by the solubi-
lity of the compounds in aqueous aggregation buffer. Similarly
of
tion process but overall
low Ffinal. Additionally, only A8 showed a significant decrease in
app which pointed to its interference in proto-fibril formation or
a-syn aggregation. A8 appear to have accelerated the fibrilliza-
b-sheet levels remained lower indicated by
K
fibrillization process.
reducing the concentration of a-syn to tip the stoichiometric ratio
From the above analysis it appears that compounds could sup-
press fibrilization to a certain extent acting as thermodynamic in-
hibitors only [32]. Here, it is important to note that we have used
only a 1:1 M ratio of protein to inhibitor, and since inhibitors could
towards inhibitor could not be practiced as a mere two-fold drop in
the concentration, lengthens the aggregation kinetics to nearly
three weeks.
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M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
Fig. 2. Molecular interaction of compounds A1, A2, A4, A8 and A9 with
a
-syn (PDB ID 1XQ8). (A) Binding interactions of compound A1 with
-syn with A8 compound. (E) A9 with C-terminus residues of
interactions are shown with yellow and red coloured dotted lines respectively. PyMOL
viewer was used for the analysis of the docked structures. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
a
-syn. (B) Molecule A2 in the binding
pocket of
a
-syn. (C) A4 in the binding pocket of
a
-syn. (D) Interaction between the essential residues of
a
a-syn. Tyr is
shown in magentas, Met in orange, Glu in green and Ser in red colours. H-bonding and
p-p
2
.3.3. Fluorescence microscopy imaging of inhibition of
a
-syn
Some studies have suggested that ThT could be knocked off in
the presence of inhibitors leading to a lower fluorescence value or
false negatives under the fluorescence microscope [34]. Hence, we
added more ThT to the mixture to ensure that any unlabelled
aggregate are also illuminated and collected the images again. The
images before (i.e. directly from the sample well) and after adding
ThT were qualitatively similar which supported our earlier obser-
vations about inhibitory potentials of compounds.
fibrillogenesis and disaggregation
Based on ThT fluorescence results, we selected A1, A2, A8 and A9
for further evaluation as they exhibited significant inhibition
(
p < 0.01). Additionally A4 was chosen based on docking results and
moderate inhibition (p < 0.05) by ThT fluorescence assay. After the
completion of the assay (ꢂ66 h) aggregated samples (control as
well as inhibited) were observed under the fluorescence micro-
scope. Since ThT was already bound to fibrils and had become
fluorescently active, no additional dye was required and the sam-
ples could be directly visualized through FITC filter. Representative
While several small molecules can inhibit
only a few have the ability to disaggregate preformed
To further test if A1, A2, A4, A8 and A9 can also disaggregate pre-
viously formed aggregates, we incubated -syn fibrillary aggregates
a-syn aggregation
a
-syn fibrils.
images are shown in Fig. 6. As expected, uninhibited
a
-syn sample
a
displayed abundant brightly illuminated fibrous structures (Fig. 6a).
In the case of A2, only a very few ThT positive aggregates were
visible. A4 and A8 also appear to be effective in lowering the
density of fluorescently illuminated aggregates but A1 and A9 were
only moderately effective. Considering the heterogeneity of the
aggregated samples it was not feasible to do a quantitative analysis
but the reduction in fluorescently labelled aggregates was clearly
visible when imaged at a lower zoom level covering a much larger
area.
(formed in the absence of ThT and any inhibitor) in the presence of
ꢁ
the compounds at 37 C for a period of 5 days in the same (1:1)
a-
syn to inhibitor molar ratio as used earlier. After adding ThT, the
samples were observed under the fluorescence microscope
through FITC filter (see Fig. 7). While the control a-syn sample was
populated with brightly illuminated fibrous aggregates expectedly,
samples incubated with A1, A2, A8 and A9 had sparsely populated,
dimly lit aggregates only indicating that these compounds were
also effective disaggregators. However, A4 was not able to dissolve
7

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
Scheme 1. Synthesis of diphenyl triazine-based compounds.
pre-formed fibrils as the corresponding images were qualitatively
2.3.4. Cytotoxicity evaluation through MTT assay
similar to the control samples.
The cytotoxicity of inihibitor compounds was determined in
mouse embryonic fibroblasts (MEF) which is a non-cancerous cell
line. The cells were grown in a media containing the compounds
Based on the above evidences we deduced that a-syn aggrega-
tion inhibition is sensitive to variation of substitution to the
diphenyltriazine moiety. We further tried to correlate the nature of
the substituent with the inhibitory potential of the compounds
A1eA9. Here, A2 appeared to be the most promising inhibitor
which not only showed the highest affinity towards a-syn upon
docking but was also the best in situ inhibitor as well as dis-
aggregator. This was probably due to higher affinity of the fused
(A1, A2, A4, A8 and A9) at concentrations of 10 mM and cell viability
was determined using MTT assay. As a control, we used DMSO
which was also used for solubilizing compounds for stock prepa-
ration. As shown in Fig. 8, compounds A1, A4, A8 and A9 did not
cause any significant toxicity to the cells even after 24 h of treat-
ment. Only A2 appeared to be mildly toxic at 10
mM. The com-
triazole-pyrimidine group towards
charge-charge interaction.
A3, A5, A6 and A7 contain methylphenyl, napthaline, dichlor-
ophenyl and nitrophenyl groups attached to the sulphonyl group,
respectively which makes them comparatively less bulky and
a
-syn perhaps due to added
pounds were further tested against human adenocarcinomic
alveolar basal epithelial (A549) cells as shown in supplementary
information (Fig. S1). Concentrations as high as 0.4 mM for all the
compounds were well tolerated with no significant cytotoxicity
except A2 which killed >60% cells at 50 mM. Thus, barring A2, which
hence the reason for their insignificant role in
a
-syn disaggregation.
incidentally seemed to be the most promising candidates based on
docking studies as well as experimental evidence, all other com-
pounds can be safely employed for further assessment of inhibitory
potential in cell culture models.
Interestingly, A8 and A9 which carry a phenylacetamide group
attached to sulphonyl at para position were very effective in
inhibiting aggregation as well as dissolving preformed aggregates.
8

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
hydrogen bond donors and acceptors, size (MW, number of rings)
and shape (ovality) descriptors, surface and volume descriptors
(hydrophobic, hydrophilic), amphiphilicity, etc were found to be in
a range favouring their ability to cross BBB for all compounds
tested.
3. Conclusion
In summary, nine new diphenyltriazine derivatives were syn-
thesized and characterized as inhibitors of a-syn fibril formation as
well as disaggregators. The compounds were first designed using
AutoDock Vina version 1.5.6 and then synthesized using convergent
organic synthetic routes. Docking studies revealed that these
compounds participate actively in non-covalent interaction with
the key residues of the
A2, A8 and A9 showed a significant lowering of the
a
-syn protein. From in-vitro ThT assay, A1,
-syn fibril
a
formation even at equimolar ratio which was further confirmed by
fluorescence microscopy. These compounds were also the top
binders predicted by the docking study and further found to be
effective disaggregators of preformed a-syn aggregates. Except A2
which was moderate cytotoxic, all the compounds were found to
have no significant effect on cellular viability. Also A4 and A8
Fig. 3.
-syn fractions eluted in PB buffer using varying NaCl concentration (B) Detection of
purified protein by Western blot using -syn specific monoclonal antibody H3C.
a-Syn purification and characterization. (A) 15% SDS-PAGE image of purified
a
appeared to ameliorate cytotoxicity associated with
aggregates.
a-syn
a
Overall, we have successfully demonstrated the utility of
diphenyl triazine hybrids as inhibitors for -syn aggregation. These
2.3.5. Effect of inhibitors on aggregated
a-synuclein cytotoxicity
a
The cytotoxic behaviour of invitro aggregated
well documented in the literature [5]. Any promising inhibitor shall
not only lower the aggregate count but also reduce the cytotoxicity
a
-syn has been
scaffolds could be further tested for efficacy in cellular as well as
in vivo models of PD treatment.
associated with aggregated
treated with monomeric
a
a
-syn. Accordingly, the MEF cells were
-syn, aggregated -syn and -syn
4. Experimental
a
a
aggregated in the presence of inhibitors. As shown in the Fig. 9,
aggregated -syn treated with MEF cells, caused a significant
decrease in cell viability (p < 0.05). The cells incubated with -syn
4.1. Docking protocol
a
a
Crystal structure of a-syn (PDB ID 1XQ8) in pdb format was
aggregated in the presence of inhibitors A1, A2 and A9 also showed
a moderate toxic effect on cells. However, the cells incubated with
downloaded from Protein Data Bank (www.rcsb.org) [40]. Auto-
Dock Vina version 1.5.6 was used to perform docking with the
ligand molecules. During the docking process, the simulation boxes
were prepared large enough to cover the interaction residues of the
protein and the ligand [26]. PyMOL visualization tool was used to
visualize the molecular interactions. Weak non-covalent in-
a
-syn aggregated in the presence of A4 and A8 exhibited no cyto-
toxicity and appeared to have a protective effect on MEF cells
against -syn aggregates. These findings indicate that A4 and A8
not only reduce the aggregation level in vitro but also ameliorate
any cytotoxicity associated with aggregated -syn and hence are
the prime candidates for further development.
a
a
teractions like
p-p
stacking interaction and H-bonding were
-syn
observed between the hydrophilic C-terminus residues of
a
and the ligand molecules lying within a range of 2.5 Å. In addition,
binding free energy was calculated on the basis of which the
designed molecules were screened.
2.3.6. Prediction of BBB permeability and drug likeness
Carrying the lead drug candidates through the bloodꢀbrain
barrier (BBB) is a major challenge and fate deciding factor for the
development of novel neurotherapeutics [35]. Various triazine
based compounds having certain similarities in their structure, size
and shape with the compounds of the present study have been
found to cross BBB. For example, BKM120 (Buparlisib) a triazine
cores structure-based brain penetrable pan-PI3K/mTOR inhibitor is
clinically used for the treatment of cancer [36]. Similarly, PQR309
4.2. Chemistry
All reagents and solvents were commercially available and used
as purchased without further purification. Melting points (mp)
were determined on a Stuart Scientific SMP1 apparatus and are
uncorrected. Proton nuclear magnetic resonance ( H NMR) spectra
were recorded on a Bruker 300 MHz FT spectrometer. Proton
1
(
Bimiralisib) is also reported to be orally available, cross the
chemical shifts are reported in ppm (TMS,
vent reference relative to TMS employed as the internal standard
(CDCl 7.26; DMSO‑d 2.54). The multiplicities of NMR signals
d 0.00) or with the sol-
bloodꢀbrain barrier, and display favorable pharmacokinetic pa-
rameters in mice, rats, and dogs [37].
3
,d
6
d
We used AdmetSAR: a wide-ranging source for calculation of
chemical ADMET properties of the compounds [38]. Most of the
compounds were found to follow the parameters such as hydrogen
bond donors less than 5, hydrogen bond acceptors less than 10,
rotatable bonds less than 10, molecular weight less than 750 Da,
and logP less than 10 (Table S1, Supporting information).
In addition, we used BBB predictor developed by Liu et al. [39] to
confirm whether a compound can cross the BBB (BBBþ) or not
are designated as s (singlet), d (doublet), dd (double doublet), t
(triplet), q (quartet), br (broad), m (multiplet, for unresolved lines).
LCMS of the compounds were carried out on applied biosystem
(absciex 2000 triple quad). Column chromatography was per-
formed on columns packed with alumina from Merck (60e120
mesh). Aluminum oxide thin-layer chromatography (TLC) cards
from Merck (aluminum oxide-precoated aluminum cards with
fluorescent indicator detectable at 254 nm) were used for TLC.
Developed plates were visualized by a Spectroline ENF 260C/FE UV
(
BBBꢀ). Lipophilicity (clogP), charges, flexibility (rotatable bonds),
9

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
Fig. 4. Time dependent aggregation kinetics for
a
-syn and
a
-syn incubated with inhibitors (A1-A9). Each panel (AeI) represents ThT fluorescence (Ex: 440 nm and Em: 485 nm)
ꢁ
from aggregation reaction of 70
mM
a
-syn in PB buffer supplemented with 10% (v/v) DMSO at 37 C in the absence (black dot) or presence of 70
mM inhibitor compounds (red
dots).Here, each data point represents ± SEM of fluorescence values from four different wells of three independent replicates. (For interpretation of the references to colour in this
figure legend, the reader is referred to the Web version of this article.)
apparatus. Organic solutions were dried over anhydrous sodium
sulfate. Evaporation of the solvents was carried out on a Büchi
rotavapor R-100 equipped with a Büchi V-100 vacuum controller.
Elemental analyses were obtained in an Elementar Vario analyser.
Elemental analyses of the compounds were found to be within
After 1 h, chloroacetic acid (1.1 g, 11.1 mol) dissolved in 30 mL
acetone was added dropwise to the reaction mixture. The reaction
mixture was allowed to reflux for 4 h at 60 C. At the end of the
reaction, the excessive acetone was removed from the reaction
vessel under vacuum, cold water was added, and the organic
compound was extracted with ethylacetate (50 mL x 3). The organic
ꢁ
±
0.4% of the theoretical values. The purity of tested compounds was
95%.
>
2 4
layer was washed with brine solution and dried over Na SO . The
crude product: 4 was purified by column chromatography using
4
.2.1. General procedure for the synthesis of compound 3 [27]
ethylacetate hexane usually 15:85.
Thiosemicarbazide (0.65 g, 7.1 mol) dissolved in 30 mL water
was put for regular stirring at room temperature. K
2
CO
3
(2 g,
4.2.3. General procedure for the synthesis of compounds A1eA9
Compound 4 (0.2 g, 0.6 mol) was dissolved in 30 mL DMSO at
room temperature. Triethylamine (0.2 g, 1.9 mol) was added to the
reaction mixture. After 1 h, the piperazine containing nucleophile
(0.68 mol) dissolved in 10 mL DMSO was added to the reaction
mixture. 0.7 g (1.9 mol) HBTU was added to the reaction and the
reaction was allowed for regular stirring at room temperature for
24 h. At the end of the reaction, the reaction mixture was poured
into ice-water and the organic phase was extracted with ethyl-
acetate (50 mL x 3). The organic layer was washed with brine so-
14.4 mol) was added to the stirring solution of thiosemicarbazide.
The reaction mixture was allowed for stirring at room temperature
for 1 h. Now ethanolic solution of benzil (1.5 g, 7.1 mol) was added
ꢁ
to it. The reaction mixture was refluxed at 80 C for 16 h. After the
completion of the reaction, the reaction mixture was acidified to pH
3
with acetic acid to obtain a yellow coloured precipitate. The
precipitate obtained was filtered and dried and was used for for-
ward reaction steps without any purification.
4.2.2. General procedure for the synthesis of compound 4
2 4
lution and dried over Na SO . The target compounds obtained were
Triethylamine (1.7 g, 16.8 mol) was added to a solution of
purified using column chromatography with eluting solvents:
compound 3 (3 g, 11.1 mol) in 30 mL acetone at room temperature.
hexane and ethylacetate usually in 20: 80 ratios.
10

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
4
.2.3.1. 1-(4-(7-Chloroquinolin-4-yl)piperazin-1-yl)-2-((5,6-
diphenyl-1,2,4-triazin-3-yl)thio)ethan-1-one (A1). Yellow solid,
ꢁ
1
yield ¼ 76% (Rf ¼ 0.7 in pure ethylacetate); m.p. 210e212 C.; H
NMR (300 MHz, CDCl
8.75 (d, J ¼ 4.8 Hz, 1H), 8.07 (d, J ¼ 1.5 Hz,
H), 7.96 (d, J ¼ 9.0 Hz, 1H), 7.62e7.30 (m, 10H), 7.26 (s, 1H), 6.86 (d,
3
) d
1
13
J ¼ 4.8 Hz, 1H), 4.40 (s, 2H), 3.98 (s, 4H), 3.26 (d, J ¼ 17.7 Hz, 4H).
NMR (75 MHz, CDCl 169.53, 166.35, 156.38, 155.87, 154.27,
51.82, 149.99, 135.26, 135.04, 134.98, 131.05, 129.82, 129.30, 128.91,
C
3
) d
1
1
28.64, 128.53, 126.68, 124.78, 121.77, 109.40, 51.96, 46.24, 42.36,
þ
38.61. LCMS: (ESI, m/z): [MþH] calcd. for C30
6
H25ClN OS 552.15;
6
found 552.9. Anal. Calcd. for C30H25ClN OS: C, 65.15; H, 4.56; Cl,
6.41; N, 15.20; O, 2.89; S, 5.80%; found C, 65.35; H, 4.36; Cl, 6.29; N,
15.33; O, 2.68; S, 5.60%.
4
[
.2.3.2. 2-((5,6-Diphenyl-1,2,4-triazin-3-yl)thio)-1-(4-(7-methyl-
1,2,4]triazolo[1,5-a]pyridin-5-yl)piperazin-1-yl)ethan-1-one (A2).
Yellow solid, yield ¼ 76% (Rf ¼ 0.7 in pure ethylacetate); m.p.
ꢁ
13
2
06e208 C.; C NMR (75 MHz, CDCl
3
) d 169.39, 166.48, 165.19,
155.96, 154.34, 154.24,149.88, 134.99, 134.92, 131.07, 129.79, 129.60,
Fig. 5. Final ThT plateau fluorescence for
a
-syn (control) and
a
-syn incubated with
129.28, 128.64, 128.53, 94.85, 47.77, 45.69, 40.89, 33.28, 25.19.
þ
inhibitors (A1-A9). Here, data represents ± SEM of fluorescence values from four
different wells after reaching plateau phase (ꢃ66 h). *** ¼ p < 0.001, ** ¼ p < 0.01 and
LCMS: (ESI, m/z): [MþH] calcd. for C28
H
N
OS 522.20; found
26
8
5
23.18. Anal. Calcd. for C28
H
26
N
8
OS: C, 64.35; H, 5.01; N, 21.44; O,
*
¼ p < 0.05, ns ¼ nonsignificant.
3.06; S, 6.13%; found C, 64.55; H, 5.31; N, 21.24; O, 3.36; S, 6.00%.
Table 2
ThT fluorescence curve derived values of t1/2, tlag, and kapp for
incubated with inhibitors.
a
-syn and a-syn
4
.2.3.3. 2-((5,6-Diphenyl-1,2,4-triazin-3-yl)thio)-1-(4-
tosylpiperazin-1-yl)ethan-1-one (A3). Yellow solid, yield ¼ 76%
app (x10 3
ꢀ
h
ꢀ1
)
ꢁ
1
Aggregation reaction
-Syn
t
1/2 (h)
t
lag (h)
K
(Rf ¼ 0.7 in pure ethylacetate); m.p. 197e199 C.; H NMR
300 MHz, CDCl
7.61 (d, J ¼ 8.1 Hz, 2H), 7.50 (d, J ¼ 6.9 Hz, 2H),
7.47e7.21 (m, 10H), 4.24 (s, 2H), 3.80e3.73 (m, 4H), 3.06 (dd,
(
3
) d
a
31.2 ± 0.6
28.2 ± 0.5
23.6 ± 1.8
29.4 ± 0.4
27.0 ± 0.2
30.0 ± 1.3
30.4 ± 1.5
29.1 ± 1.3
27.5 ± 0.6
28.1 ± 0.2
15.1 ± 0.9
15.9 ± 1.0
11.6 ± 1.4
14.1 ± 1.1
14.4 ± 0.3
16.1 ± 0.5
13.9 ± 1.4
15.3 ± 0.5
14.6 ± 0.5
14.9 ± 0.3
108.7 ± 1.8
128.1 ± 1.7
144.0 ± 15.0
118.3 ± 0.7
120.3 ± 1.2
116.2 ± 0.7
121.7 ± 5
a-Syn þ A1
a-Syn þ A2
a-Syn þ A3
a-Syn þ A4
a-Syn þ A5
a-Syn þ A6
a-Syn þ A7
a-Syn þ A8
a-Syn þ A9
13
J ¼ 27.6, 4.2 Hz, 4H), 2.41 (s, 3H). C NMR (75 MHz, CDCl
3
) d 169.35,
166.09, 155.83, 154.22, 144.15, 135.06, 134.92, 132.24, 131.06, 129.89,
129.79, 129.59, 129.31, 128.65, 128.51, 127.73, 67.08, 45.74, 33.33,
þ
21.53. LCMS: (ESI, m/z): [MþH] calcd. for C28
H N
27 5
O
3
S
2
545.16;
123.2 ± 1.9
86.6 ± 5.2
121.3 ± 3.6
found 546.0. Anal. Calcd. for C28
H
27
N
5
O
S
2
C, 61.63; H, 4.99; N,
3
12.83; O, 8.80; S, 11.75%; found C, 61.43; H, 4.79; N, 12.99; O, 8.61; S,
11.96%.
Fig. 6. Representative fluorescence microscopic images of exhibiting the inhibitory effect of compounds A1, A2, A4, A8 and A9 on
syn. (BeF)
-syn þ A1, A2, A4, A8 and A9 respectively. For all microscopic images, all imaging parameters including laser power and zoom level were kept constant. Here, Scale bar
represents 10 m.
a-syn aggregation. (A) uninhibited aggregated a-
a
m
11

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
Fig. 7. The representative fluorescence microscopic images showing the disaggregating ability of compounds A1, A2, A4, A8 and A9 towards preformed
aggregated -syn. (BeF) pre-aggregated
-syn þ A1, A2, A4, A8 and A9 respectively. For all microscopic images, all imaging parameters including laser power and zoom level were
kept constant. Here, Scale bar represents 10 m.
a-syn fibrils. (A) uninhibited
a
a
m
Fig. 8. The % cell viability of Mouse Embryonic Fibroblast cells incubated with com-
pounds (A1, A2, A4, A8 and A9) for 24 h duration. Here, 10 M concentration was used
m
for all of the compounds. Data represent ± SEM of three independent replicates. DMSO
was used as a control.
4
.2.3.4. 1-(4-((4-Bromophenyl)sulfonyl)piperazin-1-yl)-2-((5,6-
diphenyl-1,2,4-triazin-3-yl)thio)ethan-1-one (A4). Yellow solid,
ꢁ
1
yield ¼ 76% (Rf ¼ 0.7 in pure ethylacetate); m.p. 223e225 C.; H
NMR (300 MHz, CDCl 7.58 (s, 4H), 7.54e7.13 (m, 10H), 4.23 (s,
H), 3.75 (d, J ¼ 26.1 Hz, 4H), 3.09 (d, J ¼ 38.4 Hz, 4H). C NMR
75 MHz, CDCl 166.23, 155.92, 154.27, 135.06, 134.89, 134.46,
3
) d
13
2
(
Fig. 9. The % cell viability of MEF (Mouse Embryonic Fibroblast) cells incubated with
monomeric -syn, aggregated -syn (Aggr Syn) and -syn aggregated in the presence
of compounds (A1, A2, A4, A8 and A9) for 24 h duration. Data represent ±SEM of three
independent replicates. *p < 0.05 and ns nonsignificant.
3
)
d
a
a
a
1
6
C
C
32.58, 131.10, 129.80, 129.63, 129.30, 129.13, 128.71, 128.52, 128.41,
þ
7.09, 45.71, 41.67, 38.61. LCMS: (ESI, m/z): [MþH] calcd. for
27
H
24BrN
24BrN
5
O
O
3
S
S
2
609.05; found 612.0. Anal. Calcd. for
: C, 53.12; H, 3.96; Br, 13.09; N, 11.47; O, 7.86; S,
27
H
5
3
2
1
ꢁ
yield ¼ 76% (Rf ¼ 0.7 in pure ethylacetate); m.p. 231e233 C.; H
NMR (300 MHz, CDCl
8.32 (s, 1H), 7.94 (dd, J ¼ 17.1, 8.1 Hz, 3H),
.75e7.55 (m, 3H), 7.53e6.99 (m, 10H), 4.21 (s, 2H), 3.75 (dd, J ¼ 9.9,
10.50%; found C, 53.32; H, 3.76; Br, 13.29; N,11.66; O, 7.65; S, 10.71%.
3
) d
7
4
d
1
3
.8 Hz, 4H), 3.20 (dd, J ¼ 21.9, 4.8 Hz, 4H). C NMR (75 MHz, CDCl
3
)
4
2
.2.3.5. 2-((5,6-Diphenyl-1,2,4-triazin-3-yl)thio)-1-(4-(naphthalen-
-ylsulfonyl) piperazin-1-yl)ethan-1-one (A5). Yellow solid,
169.35, 166.05, 155.75, 154.17, 135.03, 134.91, 132.52, 132.20,
12

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
131.00, 129.76, 129.55, 129.43, 129.30, 129.16, 129.10, 128.63, 128.45,
Strains Escherichia Coli DH5a and Escherichia Coli Bl21 (DE3) were
1
27.99, 127.79, 122.80, 122.67, 67.08, 45.70, 41.71, 33.47. LCMS: (ESI,
purchased from New England Bio Labs, New England. Plasmid
pET21a alpha-Syn was obtained as a gift from the Michael J Fox
Foundation Hagerstown, USA.
þ
m/z): [MþH] calcd. for C31
27 5 3 2
H N O S : 581.16; found 582.0. Anal.
27 5 3 2
Calcd. for C31H N O S : C, 64.01; H, 4.68; N, 12.04; O, 8.25; S,
11.02%; found C, 64.20; H, 4.48; N, 12.25; O, 8.03; S, 11.22%.
4
.3.1. Expression and purification of
E. Coli BL21 (DE3) cells were transformed with pET21a-alpha-
synuclein plasmid [28] construct using calcium chloride
a-syn protein
4
.2.3.6. 1-(4-((2,4-Dichlorophenyl)sulfonyl)piperazin-1-yl)-2-((5,6-
diphenyl-1,2,4-triazin-3-yl)thio)ethan-1-one (A6). Yellow solid,
a
ꢁ
1
yield ¼ 76% (Rf ¼ 0.7 in pure ethylacetate); m.p. 200e202 C.; H
NMR (300 MHz, CDCl
8.04 (d, J ¼ 2.1 Hz, 2H), 7.60e7.08 (m, 11H),
.29 (s, 2H), 3.76 (s, 4H), 3.38 (d, J ¼ 37.5 Hz, 4H). C NMR (75 MHz,
CDCl 169.34, 166.37, 155.90, 154.30, 137.32, 135.01, 134.91,
33.89, 133.37, 131.77, 131.07, 130.42, 129.79, 129.57, 129.32, 128.63,
method. Transformed E. Coli BL21 (DE3) cells containing pET21a-
3
)
d
alpha-synuclein plasmid were streaked on LB agar plates contain-
ing antibiotic ampicillin (100 mg/mL). A single colony was used to
13
4
3
)
d
inoculate into 10 mL overnight cultures in LB medium with ampi-
ꢁ
1
cillin (100
m
g/mL) at 37 C, 200 rpm. The overnight culture of
þ
1
28.52, 45.84, 42.24, 38.62, 33.33. LCMS: (ESI, m/z): [MþH] calcd.
for 599.06; found 602.1. Anal. Calcd. for
: C, 54.00; H, 3.86; Cl, 11.81; N, 11.66; O, 7.99; S,
0.68%; found C, 54.19; H, 3.65; Cl, 11.62; N, 11.46; O, 7.79; S, 10.98%.
transformed E. Coli BL21 (DE3) with pET21a-alpha-synuclein was
diluted to 100-fold and induced until OD600 of 0.4e0.6 for 5 h with
1 mM of IPTG. After the completion of the incubation period, cells
27 2 5 3 2
C H23Cl N O S :
27 2 5 3 2
C H23Cl N O S
ꢁ
1
were centrifuged at 10,000 g for 30 min at 25 C. Immediately after
centrifugation, cell pellet from 1 L culture was resuspended in
100 mL osmotic shock buffer solution (30 mM Tris-HCl, 40% sucrose
and 2 mM EDTA, pH 7.2) and incubated at RT for 10 min. After
resuspension, centrifugation at 12,000g for 30 min at 4 C was done
to collect pellet. Then the collected pellet was resuspended in
4
.2.3.7. 2-((5,6-Diphenyl-1,2,4-triazin-3-yl)thio)-1-(4-((2-
nitrophenyl) sulfonyl) piperazin-1-yl)ethan-1-one (A7). Yellow solid,
ꢁ
1
ꢁ
yield ¼ 76% (Rf ¼ 0.7 in pure ethylacetate); m.p. 195e197 C.; H
NMR (300 MHz, CDCl 7.58 (s, 4H), 7.54e7.13 (m, 10H), 4.23 (s,
H), 3.75 (d, J ¼ 26.1 Hz, 4H), 3.09 (d, J ¼ 38.4 Hz, 4H). C NMR
75 MHz, CDCl 169.35, 166.26, 155.90, 154.30, 148.36, 135.07,
3
)
d
13
2
(
2
90 mL cold water with 40 mL of saturated MgCl and the solution
3
)
d
was incubated in ice for 3 min. Further, centrifugation was done at
ꢁ
134.92, 134.06, 131.73, 131.06, 130.92, 129.79, 129.55, 129.34, 128.63,
12,000 g for 30 min at 4 C. The supernatant collected after
þ
1
28.52, 124.27, 46.08, 45.69, 42.02, 33.34. LCMS: (ESI, m/z): [MþH]
centrifugation was adjusted to pH 4 using diluted HCL to precipitate
out other proteins. Again centrifugation was done at 12,000 g for
calcd. for C27
24
H N
6
O
5
S
2
: 576.12; found 576.9. Anal. Calcd. for
ꢁ
C
H N
27 24 6
O
S
5 2
: C, 56.24; H, 4.20; N, 14.57; O, 13.87; S, 11.12%; found
30 min at 4 C. After centrifugation, the collected supernatant was
C, 56.03; H, 4.40; N, 14.78; O, 13.66; S, 11.32%.
adjusted to pH 7.4 using 1 M NaOH and loaded into the DEAE-
Sepharose matrix to perform anion exchange chromatography.
Gradient elution was performed using 50 mMe500 mM NaCl,
20 mM sodium phosphate pH 8.0 to collect desired protein frac-
tions. The various fractions of eluates were pooled and then
analyzed by 15% SDS-PAGE and by Western blot using specific
4
.2.3.8. N-(4-((4-(2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)acetyl)
piperazin-1-yl)sulfonyl)phenyl)acetamide (A8). Yellow solid,
yield ¼ 76% (Rf ¼ 0.7 in pure ethylacetate); m.p. 222e224 C.; H
NMR (300 MHz, CDCl
7.97 (s, 1H), 7.67 (q, J ¼ 8.7 Hz, 4H),
.53e7.23 (m, 10H), 4.24 (s, 2H), 3.73 (d, J ¼ 14.7 Hz, 4H), 3.05 (d,
ꢁ
1
3
) d
7
antibody such as H3C (Developmental Studies Hybridoma Bank).
13
ꢁ
J ¼ 26.1 Hz, 4H), 2.19 (s, 3H). C NMR (75 MHz, CDCl
3
)
d
169.35,
The desired
a
-syn protein was further dialyzed against water at 4 C
166.17, 155.89, 154.27, 143.11, 134.95, 134.81, 131.13, 129.77, 129.62,
for 6 h and lyophilized. Lyophilized protein was further used to
perform aggregation kinetics.
1
29.29, 128.81, 128.65, 128.51, 119.50, 67.06, 46.11, 45.67, 41.66,
þ
3
5
4
3.49, 24.56. LCMS: (ESI, m/z): [MþH] calcd. for C29
28 6 4 2
H N O S :
: C, 59.17; H,
88.16; found 589.0. Anal. Calcd. for C29
H
28
N
6
O
4
S
2
4.3.2. Preparation of monomeric
The lyophilized -syn protein was dissolved in PB (20 mM So-
dium Phospahate) buffer, pH 7.4 and then adjusted to pH ~ 11 with
M NaOH solution to dissolve preformed aggregates. After
a-syn
.79; N, 14.28; O, 10.87; S, 10.89%; found: C, 59.27; H, 4.58; N, 14.49;
a
O, 10.67; S, 10.69%.
1
4
.2.3.9. N-(4-((4-(2-((5,6-diphenyl-1,2,4-triazin-3-yl)thio)acetyl)
10e15 min, protein dissolved in PB buffer was slowly adjusted to
piperazin-1-yl)sulfonyl)-2-fluorophenyl)acetamide
(A9). Yellow
pH 7.4 with 1 M HCl. Then, centrifugation was done at 100,000 g for
1 h at 4 C to remove preformed aggregate. The supernatant was
collected and filtered through a 0.22 mM filter to remove any par-
ꢁ
ꢁ
solid, yield ¼ 76% (Rf ¼ 0.7 in pure ethylacetate); m.p. 219e221 C.;
1
H NMR (300 MHz, CDCl
H), 3.77 (d, J ¼ 4.5 Hz, 4H), 3.11 (d, J ¼ 29.7 Hz, 4H), 2.23 (s, 3H).
NMR (75 MHz, CDCl 169.36, 168.71, 166.10, 155.85, 154.23,
35.04, 134.90, 131.05, 129.79, 129.56, 129.30, 128.63, 128.51, 127.47,
3
)
d
8.79 (s, 1H), 7.64e7.11 (m, 13H), 4.24 (s,
1
3
2
C
ticulate matter [41,42]. The concentration of protein was further
determined by Nanodrop by measuring the absorbance at 280 nm
3
) d
ꢀ1
ꢀ1
1
using the extinction coefficient of 5120 M cm [42].
1
24.01, 121.34, 115.83, 45.74, 41.66, 38.61, 33.49, 29.68, 24.52. LCMS:
þ
(
ESI, m/z): [MþH] calcd. for C29
H27FN
6
O
4
S
2
: 606.15; found 607.0.
4.3.3. Aggregation kinetics of
Thioflavin-T stock (5 mM) was made in PB buffer, pH 7.4 and
then filtered through 0.22 m filter. Stock of the compounds (A1-
a-syn with compounds A1eA9
6 4 2
Anal. Calcd. for C29H27FN O S : C, 57.41; H, 4.49; F, 3.13; N, 13.85; O,
10.55; S, 10.57%; found C, 57.61; H, 4.29; F, 3.13; N, 13.66; O, 10.32; S,
m
10.77%.
A9) was made in DMSO because of their insolubility in aggrega-
ꢁ
tion buffer. Aggregation of
a
-syn has been performed at 37 C in the
4.3. Biological screening of the compounds
aggregation buffer (20 mM sodium phosphate), pH 7.4. Monomeric
-syn (70 M) substituted with 20 M ThT and 0.01% Sodium Azide
a
m
m
Luria Bertani Broth, Luria Bertani Broth Agar, Sodium Dodecyl
was added in 384 wells black flat bottom plate (BRAND) with
3e4 mm glass-beads (sigma) added in each wells. The plate was
Sulfate (SDS), Cell culture dishes for adherent cells (treated surface)
and MTT reagent were purchased from Himedia Laboratories
Mumbai, India. DMEM, FBS, Penicillin-Streptomycin, Trypsin-EDTA
0.25%), phenol red and PBS were bought from Gibco. Sodium
Azide, Thioflavin-T, Isopropyl Thiogalactoside (IPTG) and DMSO
sealed with transparent film.
used as a control. For compounds, 1:1 M ratio of compounds (A1-
A9) to -syn was used for determining the inhibition of aggrega-
tion. 50 L of the sample was added per well. Total four wells have
been used for control and each inhibitor. The plate incubated in
a-syn (70 mM) with 10% DMSO was
(
a
m
were purchased from Sigma Aldrich, Bangalore, India. The bacterial
13

M. Maqbool, J. Gadhavi and P. Hivare et al.
European Journal of Medicinal Chemistry 207 (2020) 112705
Tecan Infinite M200 pro multimode plate reader and real-time
aggregation kinetics was monitored in Tecan multiplate reader by
ThT fluorescence emission at 485 nm using excitation of 440 nm
until plateau phase reached for all compounds and control. Fluo-
rescence emission of ThT was measured every 30 min intervals
with orbital shaking of 30 s before every reading. All data were
represented as mean ± standard error of mean (SEM) of four in-
dependent replicates.
compounds, laser power and fluorescence intensity has been kept
constant.
4.3.7. Cytotoxicity assay
Mouse Embryonic Fibroblasts (MEFs) cells were allowed to grow
in DMEM complete media containing 10% FBS and 1% Penicillin-
streptomycin in a humidified CO2 incubator until they reached
around 80%, confluency. After reaching confluency, the cells were
5
We further analyzed the ThT fluorescence data by fitting into a
sigmoidal curve using the given equation [33]:
trypsinized and 2e3 ꢅ 10 cells were added into 96 well trans-
ꢁ
parent clear bottom plate (SPL Lifesciences) and incubated at 37 C
in a humidified CO
media was discarded and the test compounds (A1, A2, A4, A8 and
A9) at a concentration of 10 M, monomeric -syn, aggregated
syn and aggregated -syn incubated with A1, A2, A4, A8 and A9
were added in triplicates and incubated for 24 h. Further, media
was removed and MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide) (0.5 mg/mL) made in complete me-
dia added into cells and incubated for 4 h in dark. After incubation,
the media was carefully removed, and formazan crystals were
2
incubator for 24 h. After 24 h of incubation,
y þ m t
f
f
Y ¼ y þ m t þ
i
i
½ðtꢀt1=2Þ=
ꢄ
t
1
þ eꢀ
m
a
a-
a
Here, Y is the fluorescence intensity as a function of time t, y
are the intercept of the initial baseline and plateau intensity with
the y-axis, m and m are respective slopes, which are set to zero in
our case. t is the time needed to reach half the plateau intensity.
i
and
y
f
i
f
½
t
the elongation time constant was calculated by fitting the data in
above equation for each of the four wells and then averaged to
reduce unintended bioas as explained earlier [33]. The growth of
allowed to dissolve in 100
absorbance has been measured at 570 nm in BioTek SYNERGYH1
3.04.17) microplate reader. All data were represented as
mL DMSO for 30 min. After 30 min,
synuclein fibrils was captured by kapp, (¼1/
t) the apparent rate
(
constant. tlag, time required to nuclei formation was calculated from
the intercept between the lag phase and the elongation phase
linear extrapolations. Student’s t-test was performed to see the
statistical difference considering the probability (p) value less than
mean ± standard error of mean (SEM) of three independent repli-
cates. Student’s t-test was performed to see the statistical differ-
ence considering the probability (p) value less than 0.05 using
GraphPad Prism 8.0 software.
0
.05 using GraphPad Prism 8.0 software.
Author contributions
4.3.4. Fluorescence microscopy
A 10
m
L of the
a
-syn sample (control) and
a
m
-syn incubated with
M) was transferred
MM designed and synthesized all compounds. JG and SG plan-
ned experiments for various biophysical assays. JG performed all
biophysical assays. PH performed cytotoxicity assays. MM, NH, SG
and JG wrote the manuscript.
compounds (A1-A9) mixed with ThT dye (20
to a clean glass slide and covered with a coverslip. After covered
with cover slip, extra solution has been wiped with tip of kim wipes
to prevent floating of solution. The slide was imaged by Zeiss Axio
Observer inverted fluorescence microscope using fluorescein
isothio-cyanate filter using 100X (oil immersion) objective. For
control and all compounds, laser power and fluorescence intensity
has been kept constant.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
4
(
.3.5. Disaggregation assay of
A1-A9)
To check the ability of compounds (A1-A9) to disaggregate
a-syn in the presence of compounds
Acknowledgment
previously formed fibrous species,
a
-syn incubated as a control and
MM is thankful to CSIR for financial assistance in the form of
senior research fellowship (SRF). JG is thankful DBT-JRF for Ph.D.
fellowship. PH thanks MHRD for Ph.D. Fellowship and additional
fellowship during Ph.D. Programme from IIT Gandhinagar. SG ac-
knowledges SERB, Govt. of India (CRG/2018/004960) for partial
funding support. We thank Dr. Ishan Raval for his valuable inputs
and help with manuscript editing.
ꢁ
a
-syn in the presence of the compounds at 37 C in an incubator for
a period of 5 days (A1, A2, A4, A8 and A9) in the same molar ratio
1:1) in 384 well Black flat bottom plate (BRAND) and in the
(
absence of ThT. The plate was sealed with sealing tape to prevent
evaporation. For disaggregation assay, we used 1 well for control
and each compound. Further, after incubation of 5 days, the sample
was collected, and fluorescence microscopy has been performed.
Appendix A. Supplementary data
4.3.6. Fluorescence microscopy for checking the disaggregation of
a
-syn in the presence of the compounds (A1, A2, A4, A8 and A9)
To check the disaggregation ability of the compounds (A1, A2,
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112705.
A4, A8 and A9), fluorescence microscopy has been performed.
Aggregated -syn sample (control) and aggregated -syn incubated
with compounds (A1, A2, A4, A8 and A9) were taken in individual
a
a
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