Rational design of small molecules able to inhibit α-synuclein amyloid aggregation for the treatment of Parkinson’s disease

Article abstract of DOI:10.1080/14756366.2020.1816999

Parkinson’s disease is one of the most common neurodegenerative disorders in elderly age. One of the mechanisms involved in the neurodegeneration appears related to the aggregation of the presynaptic protein alpha synuclein (α-syn) into toxic oligomers and fibrils. To date, no highly effective treatment is currently available; therefore, there is an increasing interest in the search of new therapeutic tools. The modulation of α-syn aggregation represents an emergent and promising disease-modifying strategy for reducing or blocking the neurodegenerative process. Herein, by combining in silico and in vitro screenings we initially identified 3-(cinnamylsulfanyl)-5-(4-pyridinyl)-1,2,4-triazol-4-amine (3) as α-syn aggregation inhibitor that was then considered a promising hit for the further design of a new series of small molecules. Therefore, we rationally designed new hit-derivatives that were synthesised and evaluated by biological assays. Lastly, the binding mode of the newer inhibitors was predicted by docking studies.

Full text of DOI:10.1080/14756366.2020.1816999

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Rational design of small molecules able to inhibit
α-synuclein amyloid aggregation for the treatment
of Parkinson’s disease
Serena Vittorio , Ilenia Adornato , Rosaria Gitto , Samuel Peña-Díaz ,
Salvador Ventura & Laura De Luca
To cite this article: Serena Vittorio , Ilenia Adornato , Rosaria Gitto , Samuel Peña-Díaz , Salvador
Ventura & Laura De Luca (2020) Rational design of small molecules able to inhibit α-synuclein
amyloid aggregation for the treatment of Parkinson’s disease, Journal of Enzyme Inhibition and
Medicinal Chemistry, 35:1, 1727-1735, DOI: 10.1080/14756366.2020.1816999
To link to this article: https://doi.org/10.1080/14756366.2020.1816999
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1727–1735
https://doi.org/10.1080/14756366.2020.1816999
RESEARCH PAPER
Rational design of small molecules able to inhibit a-synuclein amyloid
aggregation for the treatment of Parkinson’s disease
Serena Vittorioa , Ilenia Adornatoa, Rosaria Gittoa , Samuel Pena-Dıazb,c, Salvador Venturab,c,d and
~
ꢀ
Laura De Lucaa
b
aDepartment of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina Viale Palatucci, Messina, Italy; Institut
c
de Biotecnologia i Biomedicina, Universitat Autonoma de Barcelona, Spain; Departament de Bioquimica i Biologia Molecular, Universitat
d
Autonoma de Barcelona, Spain; ICREA, Passeig Lluis Companys 23, Barcelona, Spain
ABSTRACT
ARTICLE HISTORY
Received 7 July 2020
Revised 20 August 2020
Accepted 21 August 2020
Parkinson’s disease is one of the most common neurodegenerative disorders in elderly age. One of the
mechanisms involved in the neurodegeneration appears related to the aggregation of the presynaptic
protein alpha synuclein (a-syn) into toxic oligomers and fibrils. To date, no highly effective treatment is
currently available; therefore, there is an increasing interest in the search of new therapeutic tools. The
modulation of a-syn aggregation represents an emergent and promising disease-modifying strategy for
reducing or blocking the neurodegenerative process. Herein, by combining in silico and in vitro screenings
we initially identified 3-(cinnamylsulfanyl)-5-(4-pyridinyl)-1,2,4-triazol-4-amine (3) as a-syn aggregation
inhibitor that was then considered a promising hit for the further design of a new series of small mole-
cules. Therefore, we rationally designed new hit-derivatives that were synthesised and evaluated by bio-
logical assays. Lastly, the binding mode of the newer inhibitors was predicted by docking studies.
KEYWORDS
Parkinson’s disease; alpha-
synuclein; ligand-based
pharmacophore; aggrega-
tion inhibitors;
docking studies
GRAPHICAL ABSTRACT
1. Introduction
the central NAC domain (aa 61-95) and the C-terminal domain (aa
96-140)6. In its monomeric soluble form, a-syn assumes an alpha
helical conformation upon interaction with phospholipids,7 while
in the pathological misfolded state, it aggregates into amyloid
fibrils composed by parallel hydrogen bonded b-sheets8,9. The for-
mation of these aggregates causes cytotoxicity through lipid
membrane permeabilisation, mitochondrial damage and oxidative
stress10. Another relevant mechanism that contributes to the
propagation of neurodegeneration is the prion-like spread of
a-syn aggregates. Indeed, experimental studies revealed that the
injection of a-syn inclusions in animal’s brain promotes the forma-
tion of cellular inclusions at the injection site from where they can
spread in other brain regions11. To date, the therapies available
for the treatment of PD are addressed to reduce the motor symp-
toms and include the administration of drugs able to restore the
level of dopamine. Among them the most used is L-Dopa, which
Parkinson’s disease (PD) is a neurodegenerative disorder character-
ised by the loss of dopaminergic neurons in the substantia nigra
of the brain. These cells are involved in the production of the
neurotransmitter dopamine which regulates the muscular move-
ments1. Typical manifestations of PD include motor symptoms
due to the dopaminergic loss, like bradykinesia, rigidity, postural
instability and rest tremor2. Additionally, non-motor features such
as olfactory dysfunction, constipation, cognitive impairments,
depression and sleep disorders can occur; these further symptoms
are due to the implication of the neurodegenerative process in
other areas of the peripheral and central nervous system3. The
hallmark of PD is represented by the presence of neuronal inclu-
sions, termed Lewis Bodies, mainly composed of aggregates of
misfolded alpha synuclein (a-syn)4. a-Syn is a 140 aa presynaptic
protein which regulates the release of neurotransmitters from the
synaptic vesicles5. From a structural point of view, a-syn is organ-
acts as a prodrug being converted in dopamine in the brain1,12
.
ised in three different regions: the N-terminal domain (aa 1-60), Other dopaminergic drugs used for the treatment of PD are
CONTACT Serena Vittorio
serena.vittorio@unime.it; Laura De Luca
laura.deluca@unime.it
Department of Chemical, Biological, Pharmaceutical and
Environmental Sciences, University of Messina Viale Palatucci, 13, I-98168, Messina, Italy
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

1728
S. VITTORIO ET AL.
dopamine agonists like ropinirole or rotigotine, monoamine oxi- mixture was stirred for 15 min at room temperature for desired
dase B (MAO-B) inhibitors such as rasagiline and selegiline and
catechol-O-methyltransferase (COMT) inhibitors such as tolcapone
and entacapone which inhibit the enzymes responsible of dopa-
mine metabolism2,13. Unfortunately, the use of these drugs indu-
ces unwanted side effects such as dyskinesia, dizziness,
headaches, nausea and somnolence13. More serious problems like
hallucinations, confusion and impulse control disorders are often
associated with the assumption of dopamine agonists14.
Furthermore, these therapies lose their efficacy as the disease pro-
gresses and are unable to block or reduce the neurodegenerative
process15,16. In the last decade, several efforts have been made to
find a disease modifying strategy to halt or slow the neurodegen-
eration17. In this context, the inhibition of a-syn aggregation by
small molecules proved to be a valid approach for the develop-
ment of new therapeutics for the treatment of PD and several
inhibitors have been discovered through high-throughput screen-
compounds 5 and 7–11, whereas for desired compound 6 the
reaction time was 120 min. The resulting solid residue was washed
with water, dried and recrystallized from ethanol to provide pure
desired final pyridyl-triazole derivatives 5–11. For all synthesised
compounds the registered CAS numbers have been already
assigned; However, the synthetic procedure, chemical and struc-
tural characterisation are not available in literature with exception
of compound 5, for which the structural characterisation is an
agreement with literature22.
2.2.3.1. 3-(Phenethylthio)-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-amine
(6). CAS number: 691366-30-0. Yield 12%. Yellow solid. M.p.
168–169 ꢀC. 1H NMR (DMSO-d6): d 3.04 (t, 2H, J ¼ 7.74 Hz, CH2);
3.44 (t, 2H, J ¼ 7.74 Hz, CH2); 6.19 (s, 2H, NH2); 7.21–8.72 (m, 9H,
ArH) Anal. for C15H15N5S: C, 60.58%; H, 5.08%; N, 23.55%. Found C,
60.48%; H, 5.33%; N, 23.43%.
ing campaigns and drug repositioning18,19
.
In this work, we applied a pharmacophore-based virtual screen-
ing approach as effective tool to discover novel a-syn aggregation
inhibitors. Firstly, we developed a computational model that was
subsequently combined with in vitro experiments to test their
ability to reduce a-syn aggregation; as result we discovered a
small molecule as promising inhibitor, which was used as lead
2.2.3.2. 3-((4-Fluorobenzyl)thio)-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-
amine (7). CAS number: 575460-68-3. Yield 39%. Yellow solid.
M.p. 186–187 ꢀC. 1H NMR (DMSO-d6): d 4.44 (s, 2H, CH2); 6.21 (s,
2H, NH2); 7.13–8.71 (m, 8H, ArH). 13C NMR (DMSO-d6): d 34.0;
2
2
115.1 (d, JC-F ¼ 19.1 Hz), 115.3 (d, JC-F ¼ 19.1 Hz); 121.2; 121.3;
3
3
4
compound for the development of a further series of compounds. 131.0 (d, JC-F ¼ 8.6 Hz); 131.1 (d, JC-F ¼ 8.6 Hz); 133.8 (d, JC-
1
Then, the designed molecules were synthesised, tested in vitro
¼ 2.9 Hz) 134.0; 150.1; 152.0; 154.5; 161.4 (d, JC-F ¼ 243.2 Hz).
F
and studied to decipher the putative binding mode by molecular
docking simulation.
Anal. for C14H12FN5S: C, 55.80%; H, 4.01%; N, 23.24%. Found C,
55.68%; H, 4.31%; N, 23.44%.
2.2.3.3. 3-((3-Fluorobenzyl)thio)-5-(pyridin-4-yl)-4H-1,2,4-triazol–4-
amine (8). CAS number: 674812-86-3. Yield 52%. Yellow solid.
M.p. 164–165 ꢀC. 1H NMR (DMSO-d6): d 4.45 (s, 2H, CH2); 6.25 (s,
2H, NH2); 7.06–8.72 (m, 8H, ArH); 13C NMR (DMSO-d6): d 34.6;
2. Materials and Methods
2.1. Pharmacophore modelling and virtual screening
LigandScout V4.420 was used for the pharmacophore generation
and the virtual screening experiments. Three small molecules able
to bind to the N-terminal region of a-syn have been selected from
literature21 and used as training set. A shared-feature pharmaco-
phore model was created applying the default settings.
2
2
114.6 (d, JC-F ¼ 20.9 Hz), 116.3 (d, JC-F ¼ 21.0 Hz); 121.7; 121.8;
4
3
3
125.6 (d, JC-F ¼ 2.7 Hz); 130.8 (d, JC-F ¼ 8.2 Hz); 134.4, 141.0 (d, JC-
1
¼ 8.2 Hz); 150.5; 152.5; 154.8; 162.4 (d, JC-F ¼ 243.4 Hz). Anal. for
F
C14H12FN5S: C, 55.80%; H, 4.01%; N, 23.24%. Found C, 55.89%; H,
4.00%; N, 23.15%.
All virtual screening runs were performed by setting the option
“Get best matching conformation” as retrieval mode.
2.2.3.4. 3-((4-Methylbenzyl)thio)-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-
amine (9). CAS number: 676147-32-3. Yield 13%. Yellow solid.
M.p. 180–181 ꢀC 1H NMR (DMSO-d6): d 2.25 (s, 3H, CH3); 4.40 (s,
2.2. Chemistry
All reagents were used without further purification and bought 2H, CH2); 6.19 (s, 2H, NH2); 7.10–8.71 (m, 8H, ArH). 13C NMR
from common commercial suppliers. Melting points were deter-
mined on a Buchi B-545 apparatus (BUCHILabortechnik AG Flawil,
Switzerland). By combustion analysis (C, H, N) carried out on a
Carlo Erba Model1106-Elemental Analyser we determined the pur-
ity of synthesised compounds; the results confirmed a 95% pur-
ity. Merck Silica Gel 60 F254 plates were used for analytical TLC
(Merck KGaA, Darmstadt, Germany). For detection, iodine vapour
and UV light (254 nm) were used. 1H and 13C NMR spectra were
(DMSO-d6): d 21.0; 35.2; 110.0; 121.7; 121.8; 129.3; 129.6 134.4;
134.7; 137.1; 141.0; 150.4; 150.7; 152.4; 155.1. Anal. for C15H15N5S:
C, 60.58%; H, 5.08%; N, 23.55%. Found C, 60.23%; H, 5.26%;
N, 23.40%.
2.2.3.5. 3-((3-Methylbenzyl)thio)-5-(pyridin-4-yl)-4H-1,2,4-triazol-4-
amine (10). CAS number: 676548-28-0. Yield 16%. Whitish solid.
M.p 175–176 ꢀC; 1H NMR (DMSO-d6): d 2.26 (s, 3H, CH3); 4.40 (s,
2H, CH2); 6.19 (s, 2H, NH2); 7.07–8.71 (m, 8H, ArH). 13C NMR
(DMSO-d6): d 21.3; 35.4; 121.7; 126.7; 128.5; 128.8; 130.0; 134.4;
137.6; 138.0; 150.6; 152.4; 155.1. Anal. for C15H15N5S: C, 60.58%; H,
5.08%; N, 23.55%. Found C, 60.70%; H, 5.01%; N, 23.32%.
measured in dimethylsulfoxide-d6 (DMSO-d6) with
a Varian
Gemini 500 spectrometer (Varian Inc. Palo Alto, California USA);
chemical shifts are expressed in d (ppm).
2.2.3. General procedure for the synthesis of pyridinyl-triazole
derivatives 5-11
The 4-amino-5–(4-pyridinyl)-4H-1,2,4-triazole-3-thiol 12 (200 mg,
1,03 mmol) was dissolved in a mixture of NaOH (41 mg, 1,03 mmol)
and MeOH (5 ml); after complete dissolution, the suitable benzyl
2.2.3.6. 3-((4-Chlorobenzyl)thio)-5-(pyridin-4-yl)-4H-1,2,4-triazol–4-
amine (11). CAS number: 901093-20-7. Yield 39%. Yellow solid.
M.p 198–199 ꢀC; 1H NMR (DMSO-d6): d 4.44 (s, 2H, CH2); 6.22 (s,
2H, NH2); 7.37–8.70 (m, 8H, ArH). 13C NMR (DMSO-d6): d 34.4;
bromide derivative (194,7 mg, 1,03 mmol) was added. The reaction 121.7; 121.8; 128.61; 129.0; 131.2; 131.5; 132.4; 134.4; 137.2; 150.4;

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1729
150.7; 152.5; 154.8. Anal. for C14H12ClN5S: C, 52.91%; H, 3.81%; N, 10 protein ꢁ ligand binding poses for each compound by using
23.04%. Found C, 52.66%; H, 3.78%; N, 23.36%.
the default settings. The highest scored docking pose was
chosen for analysis and representation. PyMOL software was used
to visualise docking results while the analysis of the putative
ligand–protein interactions was performed by Discovery Studio
Visualiser27.
2.2. 2.3. Alpha synuclein aggregation and inhibition
in vitro assays
WT a-Syn was expressed and purified as previously described23.
The resultant protein was kept lyophilised at ꢁ80 ꢀC. Lyophilised
protein was delicately resuspended in sterile PBS 1X and filtered
through 0.22-mm membrane to eliminate small aggregates.
Aggregation assays were performed as previously described for
ZPD-2 and SynuClean-D, the latter used as reference compound
for kinetic studies16,24. Briefly, 70 mM of a-Syn, in a final volume of
150 mL, was placed in a sealed 96-well plate, which also contains
40 lM Th-T in PBS 1X, a 1/8” diameter Teflon polyball (Polysciences
Europe GmbH, Eppelheim, Germany) and 100 lM of the different
compounds or DMSO (in control samples). The plates were fixed
in an orbital shaker Max-Q 4000 (ThermoScientific, Waltham,
Massachusetts, USA) and incubated at 37 ꢀC and 100 rpm. Th-T
fluorescence emission was measured every 2 h in a Victor3.0
Multilabel Reader (PerkinElmer, Waltham, Massachusetts, USA) by
exciting through a 430–450 nm filter and collecting with a 480-
510 filter. Triplicates were performed and the kinetics were fitted
with the following equation:
3. Results and discussion
3.1. Pharmacophore model generation, virtual screening and
preliminary biological assay
With the aim to find new molecules able to inhibit a-syn aggrega-
tion, we used a computational approach to generate a ligand-
based pharmacophore model by means of the software
LigandScout20.
To build the model, we used a training set (TS) including a-syn
aggregation inhibitors that target the N-terminal portion of the
protein. Specifically, we selected three small molecules from litera-
ture21 belonging to different chemotypes as polyphenolic and
non-polyphenolic inhibitors (Table 1) for which the binding inter-
action to N-terminal portion of a-syn is corroborated by experi-
mental data.
In particular, we considered only the common features for all
the selected molecules of the TS for the model construction gen-
erating shared feature pharmacophore models.
As result, we obtained ten pharmacophore hypotheses and
selected the hypothesis possessing the highest score to perform
virtual screening studies. As displayed in Figure 1, the above-men-
tioned pharmacophore model consisted of five features: (i) two
hydrogen bond acceptors, (ii) two hydrophobic features, (iii) one
aromatic feature and 45 excluded volumes as forbidden areas.
1
/¼ 1 ꢁ
(1)
kat
ð
e
Þ
ꢁ 1 þ 1
kb
In which kb and ka represent the homogeneous nucleation rate
constant and the secondary rate constant (it means, fibril elong-
ation and secondary nucleation), respectively25.
Light-scattering measurements were performed in
a Cary
Eclipse Fluorescence Spectrophotometer (Agilent, Santa Clara,
California, USA). 80 mL of end-point aggregates was placed into a
quartz cuvette and excited at 300 nm, the 90ꢀ emission was thus
collected between 280 and 360 nm.
For transmission electron microscopy (TEM) assays, endpoint
treated and untreated aggregates diluted 1:10 in PBS 1X. Samples
were then softly sonicated for 5 min and 5 mL of the resultant mix-
ture were placed on a carbon-coated copper grid for 5 min. The
grids were dried with filter paper to remove the excess and
washed twice in miliQ water, whose excess was also removed.
Finally, 5 lL of 2% (w/v) uranyl acetate solution was added and
left incubate for 2 min. As previously, the excess of uranyl acetate
was removed, and grids were left to air-dry for 10 min. Images
were obtained using a Transmission Electron Microscopy Jeol
1400 (Peabody, Massachusetts, USA) operating at an accelerating
voltage of 120 kV. A minimum of 30 fields were screened per sam-
ple, in order to collect representative images.
Table 1. Chemical structures of the a-syn aggregation inhibitors used as train-
ing set (TS).
NAME
STRUCTURE
Congo Red
2.4. Docking studies
Lacmoid
Docking studies were performed by Autodock4.2 software by
using the solid-state NMR of alpha-synuclein fibrils retrieved from
RCSB Protein Data Bank (PDB code 2N0A). The structure is charac-
terised by a central part with a greek-key topology and terminal
flexible loops. A grid box with dimension of 126 ꢂ 126 ꢂ 126 Å3
and centre x ¼ 97.487, y ¼ 148.695 and z ¼ ꢁ34,111, was applied
in order to include the aminoacid residues of the N-terminal
region discarding the ones present in the unstructured flexible
loops. Ligand structures were constructed by Vega ZZ software
and energy minimised by following a conjugate gradient mini-
misation by AMMP calculation as implemented in VEGAZZ pro-
gram26. The Lamarckian Genetic Algorithm was used to calculate
Rosmarinic
Acid

1730
S. VITTORIO ET AL.
Figure 1. (A) Best scored ligand-based pharmacophore model constituted by two hydrogen bond acceptors (red spheres), two hydrophobic features (yellow spheres),
one aromatic feature (blue circle). Forty-five excluded volumes are represented by grey spheres. (B) TS molecules aligned with the highest scored pharmaco-
phore model.
Figure 2. (A) Chemical structure of compounds 1a-c. (B) Compound 1a aligned to the pharmacophore model. Compound 1a is represented by grey sticks.
The obtained pharmacophore model was used as a query to yl)ethenone (1a) displaying the highest pharmacophore fit-score
screen two three-dimensional databases containing small mole- as representative inhibitor filtered through virtual screening on
cules belonging to different structural chemotypes: (i) the in-house CHIME database (Figure 2(B)).
3 D database CHIME collecting 1329 molecules designed and syn-
The virtual screening on MyriaScreen Diversity Library II
thesised by our group over the years, (ii) the MyriaScreen resulted in 113 hits (see supporting material). Among them, we
Diversity Library II, consisting of 10,000 compounds with druglike selected three compounds (Figure 3.) such as 3-[5-[(4-methoxy-
properties
services/high-throughput-screening/screening-compounds.html).
(https://www.sigmaaldrich.com/chemistry/chemistry- phenyl)methylsulfanyl]-4-methyl-1,2,4-triazol-3-yl]pyridine (2), 3-
(cinnamylsulfanyl)-5–(4-pyridinyl)-1,2,4-triazol-4-amine (3) and
From CHIME, the virtual screening led to the identification of 3-(3-chloro-4-fluoro-anilino)-1-(2-naphthyl)propan-1-one (4); the
three hits 1a–c (Figure 2(A)). Considering that these molecules selection of the compounds 2–4 was made basing on the
shared a very similar structure, we decided to select only 2-[4-[(4- pharmacophore-fit score value (>57) and the commercial avail-
fluorophenyl)methyl]-1-piperidyl]-1-(6-methoxy-1H-indol-3-
ability from the supplier.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1731
All selected compounds 1–4 proved to respect the Lipinski’s
rule and no PAINS were in silico predicted by the online tool
SwissAdme platform [http://www.swissadme.ch/]. To evaluate the
ability to inhibit a-syn aggregation, we have got compounds 1–4
as follows. Compound 1 was readily synthesised in our laboratory
following a previously optimised synthetic approach;28 while the
three commercially available compounds 2–4 were purchased
from Sigma Aldrich as supplier of bioactive molecules [https://
www.sigmaaldrich.com/italy.html].
Therefore, this first screening highlighted that the best in vitro
results were obtained for compound 3. This compound is charac-
terised by the presence of a pyridinyl-triazole moiety similarly to
compound 2, in which the nitrogen of the triazole ring presents a
methyl substituent while an amino group is present in compound
3 in the same position. Considering the obtained results, we can
speculate that the amino group could establish fundamental inter-
action with the protein important for the inhibition of a-syn
aggregation.
The anti-aggregation potential of selected compounds (1–4)
was evaluated by a robust protocol previously applied in the iden-
tification of strong inhibitors of a-syn aggregation16,24 like
SynuClean-D, a well-known inhibitor of a-synuclein (a-Syn) aggre-
gation24 that was used as reference compound in the same test.
The presence of 100 mM of the compounds during the incubation
of 70 mM of a-Syn, reduced Thioflavin-T (Th-T) fluorescence emis-
sion up to a 10, 26, 31 and 10% for compound 1, compound 2,
compound 3 and compound 4, respectively. As result, all tested
compounds impacted the kinetic constants (see Figure 4(A)).
Particularly, the compound 2 reduced the homogeneous nucle-
ation rate constant (kb ¼ 0.01591) when compared to untreated
sample (kb ¼ 0.03234). In contrast, compound 3 reduced the auto-
catalytic rate constant (ka ¼ 0.1892 hꢁ1), compared to the control
(ka ¼ 0.2037 hꢁ1). The compounds 1 and 4 did not impact signifi-
cantly the aggregation rate constants. To confirm these results
light-scattering measurements at 300 nm were performed at the
end-point of the reaction, thus revealing a reduction of 13 and
43% of aggregates in the presence of compounds 2 and 3,
respectively.
3.2. Design and synthesis of new small molecules as derivatives
of compound 3
Based on the above reported results of biological screening, we
chose compound 3 as promising lead compound for the rational
design of a further series of pyridinyl-triazole derivatives 5–11
(Scheme 1) for which a very simple synthetic approach might be
carried out.
In particular, we chose to maintain the pyridinyl-triazole moiety
of inhibitor 3 and reduce the length of the linker between the sul-
phur atom and the phenyl ring. Moreover, we planned to extend
the series by introducing selected substituents in meta and in
para positions of phenyl ring. In detail, we introduced F, Cl or CH3
substituents in order to preliminary probe the influence of elec-
tron withdrawing group (EWG) or electron donating group (EDG)
on the aggregation of a-syn. For each designed derivative we
evaluated the adherence to the Lipinski’s rule and the absence of
PAINS in silico by using SwissADME. Scheme 2 shows the syn-
thetic procedure to obtain compounds 5-11 starting from the 4-
amino-5–(4-pyridinyl)-4H-1,2,4-triazole-3-thiol 12, that was coupled
with the appropriate benzyl bromide in alkaline medium at room
temperature.
To further assess the impact of the best candidate 3 on a-syn
aggregation, end-point samples were analysed by transmission
electron microscopy (TEM). Notably, TEM images certified a signifi-
cant reduction in the amount of fibrillar material in the presence
of compound 3 (Figure 4(D)), when compared to untreated sam-
ples (Figure 4(C)).
Figure 3. (A) Chemical structures of compounds 2, 3 and 4. (B) Alignment of each of the reported hits (represented by pink (2), orange (3) and yellow (4) sticks) with
the pharmacophore model.

1732
S. VITTORIO ET AL.
Figure 4. Inhibitory in vitro characterisation of compounds 1 to 4. (A) Aggregation kinetic of a-syn in absence (black) and presence of 100 mM of Compound 1 (red),
compound 2 (green), compound 3 (blue), compound 4 (orange) and SynuClean-D (grey), followed by Th-T fluorescence emission. (B) Light-scattering measurements at
300 nm in the absence (black) and presence of 100 mM of Compound 1 (red), compound 2 (green), compound 3 (blue) and compound 4 (orange). (C and D)
Representative TEM images of untreated (C) and compound 3 treated samples (D). Th-T fluorescence is plotted as normalised means. Final points were obtained at
ꢃꢃ
ꢃꢃꢃ
48 h. Error bars are represented as SE of mean values;
p < 0.01 and
p < 0.001.
R
n
1
2
i
5
6
7
H
H
4-F
1
12
5-11
3-F
8
9
10
11
1
1
1
1
Scheme 2. Reagents and conditions: i) Ar(CH2)nBr, NaOH, MeOH, rt.
4-CH3
3-CH3
4-Cl
5-11
described above. Incubation of 70 mM a-Syn in the presence of
100 mM of the different compounds, followed by Th-T fluorescence
measurements at the end of the reaction indicated that 5, 8, 9
and 11 could reduce the aggregation up to a 33, 15, 19 and 29%,
respectively (Figure 5(A)). Light-scattering measurements at
300 nm indicated that 5, 6, 7, 8, 9 and 10 reduced the amount of
aggregates up to a 23, 16, 26, 14, 24, 17%, respectively (Figure
5(B)). However, TEM analysis indicated that only 5 (Figure 5(E)), 8
(Figure 5(F)), 9 (Figure 5(G)) and 11 (Figure 5(H)) reduced the
Scheme 1. Designed pyridinyltriazole derivatives 5–11 inspired by compound 3.
3.3. Screening of the activity of the new derivatives 5–11
The synthesised derivatives were tested in order to study their
ability to reduce a-syn aggregation in vitro by using the method

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1733
Figure 5. Inhibitory in vitro characterisation of tested compounds 5–11. (A and B) Aggregation kinetics of a-syn in the absence (black) and presence of 100 mM of
compounds 5 (red), 6 (green), 7 (violet), 8 (orange), 9 (blue), 10 (brown), 11 (yellow) and SynuClean-D (grey), followed by Th-T fluorescence emission. (C) Light-scatter-
ing measurements at 300 nm in the absence (black) and presence of 100 mM of compounds 5 (red), 6 (green), 7 (violet), 8 (orange), 9 (blue), 10 (brown) and 11 (yel-
low). (D to G) Representative TEM images of untreated (D) and 5 (E), 8 (F), 9 (G) and 11 (H) treated samples. Th-T fluorescence is plotted as normalised means. Final
ꢃꢃ
ꢃꢃꢃ
p < 0.001.
points were obtained at 48h. Error bars are represented as SE of mean values;
p < 0.01 and
Figure 6. Binding site of a-syn identified through docking studies for this class
of inhibitors. The image is created by PyMOL software (https://pymol.org).
Figure 7. Plausible binding mode of compound 3 (pink stick). The interacting
residues of the binding site are represented as light blues stick. The image is cre-
ated by PyMOL software (https://pymol.org).
number of amyloid fibrils when compared to control samples
(Figure 5(D)), in good agreement with the kinetic analysis.
Considering that in our above reported molecular modelling
studies we selected polyphenolic and non-polyphenolic inhibitors
(see Table 1) that bind the N-terminal region of a-syn, we hypoth-
esised that also derivatives 3, 5, 8, 9 and 11 could interact with
same portion of the protein. For this reason, we defined this
region as the search space for the docking simulation (see
Material and Methods section). The docking results confirmed that
all the tested compounds bind in the same site of a-syn located
between the N-terminal and the NAC domains of the protein and
lined by Ala53, Val55, Thr59, Glu61, Thr72, Gly73 and Val74
(Figure 6).
3.4. Docking studies
In order to ascertain the binding mode of pyridinyl-triazole deriva-
tives 3, 5, 8, 9 and 11 that demonstrated the ability to reduce
a-syn aggregation in vitro, we carried out molecular docking simu-
lation by means of Autodock 4.2 suite;29 the computational stud-
ies were based on NMR structure of a-syn (PDB code 2N0A) as
structural coordinates30.

1734
S. VITTORIO ET AL.
Figure 8. Plausible binding modes for compound 5 (magenta stick, panel A), 8 (orange stick, panel B), 9 (green stick, panel C) and 11 (cyan stick, panel D). The inter-
acting residues of the binding site are represented as light blues stick. The images are created by PyMOL software (https://pymol.org).
Notably, in a recent work, Pujols and co-workers identified the of Glu61. Moreover, for inhibitors 5, 8, 9 a pi-anion interaction
same portion of the protein as binding site of their inhibitor 2-
hydroxy-5-nitro-6-(3-nitrophenyl)-4-(trifluoromethyl)nicotinonitrile
through a induced-fit docking simulation24; therefore, these evi-
dences further supported the hypothesis that this region could
represent a binding site for small molecules on a-syn fibrils.
In detail, compound 3 could bind to a-syn by establishing a
salt bridge between the amino group and Glu61 and p-anionic
interaction between the pyridine ring and Glu61B. The cinnamyl
moiety is oriented towards Ala53, Gly73 and Val74 and might
engage hydrophobic interaction with Ala53. Furthermore, van der
Waals interactions with Val55, Thr59, Thr72, Gly73 and Val74 could
be observed (Figure 7).
In Figure 8, the binding mode of compounds 5, 8, 9 and 11 is
displayed. The new derivatives showed a slight different binding
orientation in comparison to the parent compound 3, probably
due to the shorter and more flexible linker between the sulphur
atom and the aromatic ring.
was observed between the pyridine ring and Glu61B.
Additionally, compound 5 might form a hydrogen bond with
Thr72C through the sulphur atom and pi-sigma interaction with
Thr72C (Figure 8, Panel A). Concerning compound 8, it might
establish a halogen bond between the fluorine atom and Thr72D
(Figure 8, Panel B). Instead, 9 could engage pi-sigma interaction
with Thr72C (Figure 8, Panel C). Finally, compound 11 might
establish van der Waals interactions with Val55C (Figure 8,
Panel D).
4. Conclusion
The search for a cure of PD represents an important challenge in
the pharmaceutical research field. The inhibition of a-syn aggrega-
tion has emerged as promising new therapeutic strategy for the
treatment of PD. In this work, we described the generation of a
ligand-based pharmacophore model, which was used as query to
In particular, all the inhibitors could interact with a-syn by a
crucial salt bridge between the amino group and the side chain screen two chemical libraries. The hits selected from the virtual

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1735
screening were tested in vitro to probe their ability to inhibit 12. Haddad F, Sawalha M, Khawaja Y, et al. Dopamine and levo-
a-syn aggregation, resulting in the identification of the new hit 3.
Structural modifications were carried out on compound 3 obtain-
ing four interesting derivatives. Finally, the binding mode of this
new class of inhibitors on a-syn was investigated by molecular
docking simulation. All the information gained from these studies
could be useful for the design of novel inhibitors a-syn aggrega-
tion inhibitors bearing a pyridinyl-triazole scaffold.
dopa prodrugs for the treatment of Parkinson’s disease.
Molecules 2017;23: 40.
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neuroprotective compounds for Parkinson’s disease. Curr
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Disclosure statement
The authors declare that there is no conflict of interest.
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16. Pena-Dıaz S, Pujols J, Conde-Gimenez M, et al. ZPD-2, a
small compound that inhibits a-synuclein amyloid aggrega-
tion and its seeded polymerization. Front Mol Neurosci
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Funding
The authors thank the “Programma Operativo Nazionale Ricerca &
Innovazione 2014-2020, Azione I(0).1 “Dottorati Innovativi con car-
atterizzazione industriale” XXXIII cycle, for financial support in
form of a PhD scholarship “DOT1314952” granted to SV.
17. Singh SK, Dutta A, Modi G. a-Synuclein aggregation modula-
tion: an emerging approach for the treatment of Parkinson’s
disease. Future Med Chem 2017;9:1039–53.
18. Shihabuddin LS, Brundin P, Greenamyre JT, et al. New fron-
tiers in Parkinson’s disease: from genetics to the clinic. J
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Serena Vittorio
Rosaria Gitto
Laura De Luca
http://orcid.org/0000-0001-6092-5011
http://orcid.org/0000-0003-0002-2253
http://orcid.org/0000-0003-0614-5713
clein for PD therapeutics:
Biomolecules 2020;10:391.
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pursuit on all fronts.
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