ISSN 0006-2979, Biochemistry (Moscow), 2026, Vol. 91, No. 5, pp. 789-799 © Pleiades Publishing, Ltd., 2026.
789
A Conjugate of Aminoadamantane
and Tetrahydro-γ-Carboline Inhibits
Accumulation of Mutant α-Synuclein A53T
in the Cellular Model of Proteinopathy
Marina V. Burak
1
, Nadezhda E. Pukaeva
1
, Victoria S. Kryshkova
1
,
Olga A. Kukharskaya
1
, Maya R. Nazdracheva
1
, Sergey A. Pukhov
1
,
Kirill A. Rachenkov
1
, Alla V. Stavrovskaya
2
, Vladimir P. Fisenko
3
,
Michail S. Kukharsky
1,a
*, and Sergei O. Bachurin
1,b
*
1
Institute of Physiologically Active Compounds, Federal Research Center of Problems
of Chemical Physics and Medicinal Chemistry, Russian Academy of Sciences,
142432 Chernogolovka, Moscow Region, Russia
2
Russian Center of Neurology and Neurosciences,
Ministry of Science and Higher Education of the Russian Federation, 125367 Moscow, Russia
3
First Moscow State Medical University (Sechenov University),
Ministry of Health of the Russian Federation, 119048 Moscow, Russia
a
e-mail: kukharskym@gmail.com
b
e-mail: bachurin@ipac.ac.ru
Received January 14, 2026
Revised March 10, 2026
Accepted March 11, 2026
Abstract— Pathological aggregation of α-synuclein is a key event in the development of synucleinopathies,
such as Parkinson’s disease and Lewy body dementia. Currently, no effective disease-modifying therapy
is available, necessitating the search for new therapeutic agents. One promising strategy involves the use
of low-molecular-weight compounds capable of inhibiting the formation of toxic protein aggregates. This
study evaluates the anti-aggregation properties of EC3222x, a conjugate of pharmacophoric fragments of
amantadine and a fluorinated derivative of tetrahydro-γ-carboline. α-Synucleinopathy was modeled in the
SH-SY5Y neuroblastoma cell line by transfection with a plasmid vector encoding the mutant human α-sy-
nuclein A53T protein. EC3222x at a concentration of 1 µM reduced the number of cells with α-synuclein
A53T aggregates. Its efficacy was comparable to that of SynuClean-D and Buntanetap, known inhibitors of
α-synuclein aggregation. Treatment with EC3222x reduced both the level of diffusely distributed intracellular
α-synuclein and the formation of mature fibrillar aggregates and large aggresomes. Importantly, EC3222x
did not affect the accumulation of another aggregation-prone protein, TDP-43, in a similar cellular model,
indicating its specificity for α-synuclein. These findings suggest that EC3222x may represent a promising
candidate for the development of therapeutic agents targeting synucleinopathies.
DOI: 10.1134/S0006297926600079
Keywords: protein aggregation, α-synuclein, neurodegenerative diseases, cellular models, protein aggregation
inhibitors, neuroprotective agents
* To whom correspondence should be addressed.
INTRODUCTION
Neurodegenerative diseases (NDDs) are a hetero-
geneous group of disorders characterized by patho-
logical protein aggregation in the nervous system,
leading to neuronal death. Synucleinopathies are a
subset of NDDs characterized by accumulation of in-
soluble α-synuclein in neurons and glial cells as cy-
toplasmic inclusions – Lewy bodies and Lewy neur-
ites [1]. This group includes Parkinson’s disease (PD),
BURAK et al.790
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026
Lewy body dementia, and multiple system atrophy.
Additionally, α-synuclein aggregates are found in the
senile plaques in Alzheimer’s disease (AD) [2-4].
Under normal conditions, α-synuclein is involved
in neuronal signaling by regulating release of neu-
rotransmitters, primarily dopamine, in presynaptic
terminals. Point mutations altering the amino acid se-
quence affect three-dimensional structure of the pro-
tein, thus accelerating oligomerization and promoting
formation of cytotoxic insoluble inclusions (aggre-
gates). Accumulation of the aggregated protein in the
cytoplasm disrupts its normal synaptic function. Fur-
thermore, oligomeric forms interact with membranes
and other proteins, engaging in metabolic processes
and exerting toxic effects on neurons [4, 5]. Several
known mutations (A18T, A29S, A30P, E46K, H50Q,
G51D, A53T, A53E) are associated with familial forms
of PD and with Lewy body dementia [6-8]. The A53T
mutant variant of α-synuclein (alanine to threonine
substitution at position 53) exhibits high propensity
for aggregation and forms fibrils faster than the wild-
type protein [9]. This mutation leads to the earlier on-
set and more severe progression ofPD [10]. Currently,
PD treatment is symptomatic and aimed at improving
quality of life of the patients [11]. Identifying ways to
influence pathogenetic mechanisms associated with
the onset and progression of the disease is essential
for developing effective therapies for PD [12].
One promising strategy for treating synucle-
inopathies is inhibiting formation and/or removal
of the already formed aggregates by binding them
with low-molecular-weight chemical compounds [13].
Recent studies aimed at finding α-synuclein aggre-
gation inhibitors have identified and characterized
compounds from various classes with this effect [14,
15]. Polyphenols, such as curcumin [16] and epigal-
locatechin-3-gallate [17], as well as quinoline and in-
dole alkaloids [18, 19] exhibit anti-aggregation prop-
erties against α-synuclein. Among the most studied
blockers of α-synuclein aggregation are SynuClean-D
and Buntanetap. SynuClean-D (SC-D, 5-nitro-6-(3-nitro-
phenyl)-2-oxo-4-(trifluoromethyl)-1H-pyridine-3-carbo-
nitrile) is a small molecule with anti-aggregation and
neuroprotective activity in PD models [20]. SC-D re-
duces oligomerization and aggregate formation, pro-
moting their disruption, and decreases intracellular
accumulation of phosphorylated α-synuclein forms
[21]. This compound inhibits aggregation of both
wild-type α-synuclein and its mutant variants, such
as A30P and H50Q. SC-D demonstrated efficacy in the
Caenorhabditis elegans PD model, where it reduced
α-synuclein aggregation, protected neurons from de-
generation, and improved animal motility [20].
Buntanetap (Bun, Posiphen, [(3aS,8bR)-3,4,8b-
trimethyl-2,3a-dihydro-1H-pyrrolo[2,3-b]indol-7-yl]
N-phenylcarbamate) acts by inhibiting α-synuclein
translation: it increases affinity of the regulatory
protein IRP1 (iron regulatory protein 1) for the IRE
(iron-responsive element) sequence in the 5′-un-
translated region of the mRNA encoding α-synuclein.
Thus, Bun prevents mRNA association with the ribo-
some and suppresses protein synthesis [22-24]. Bun
has been shown to reduce α-synuclein levels in the
human neuroblastoma cell lines and primary rodent
neurons [22, 24, 25]. Additionally, Bun reduces the
levels of other neurotoxic proteins, such as β-amy-
loid and tau protein [26, 27]. Currently, Buntanetap is
undergoing clinical trials as a treatment for AD and
PD (CTID: NCT02925650, NCT04524351).
Further search for new α-synuclein aggregation
inhibitors is necessary to obtain more effective and
selective compounds with detailed characterization
of their effects on various stages of the aggregation
process and different pathogenic protein variants,
which would facilitate development of therapies tar-
geting specific diseases.
The compound EC3222x, investigated in this
study, is a conjugate of the pharmacophoric fragments
of amantadine and a fluorinated derivative of tetra-
hydro-γ-carboline. The drug amantadine can interact
with a wide range of biological targets, including
glial cell line-derived neurotrophic factor, phosphodi-
esterase 1 (PDE1), nicotinic receptors, N-methyl-D-as-
partate (NMDA) receptors, potassium channels, and
others [28]. Through these interactions, amantadine
exhibits neuroprotective and anti-Parkinsonian ef-
fects [29]. Gamma-carbolines are nitrogen-containing
heterocyclic compounds whose neuroprotective ef-
fects have been demonstrated relatively recently [30].
In particular, they have been shown to reduce ag-
gregation levels and activate protective mechanisms
involved in the degradation of aggregated protein
species [31]. The tetrahydro-γ-carboline derivative
Dimebon has demonstrated efficacy in several in vitro
and in vivo models of NDDs [32-37]. The compound
EC3222x, in previous studies, has shown the ability to
block two NMDA receptor binding sites at micromo-
lar concentrations, inhibit acetyl- and butyryl-cholin-
esterases, potentiate microtubule assembly, and exert
mitoprotective effects [38]. In this regard, this sub-
stance is considered a lead compound in the series of
conjugated structures with potential neuroprotective
activity. In the present work, we studied the effect of
EC3222x on accumulation and aggregation of the mu-
tant human α-synuclein A53T protein in the SH-SY5Y
neuroblastoma cell line.
MATERIALS AND METHODS
Compounds. Compound EC3222x was synthesized
and described previously [38]. SynuClean-D (5-nitro- 6-
EC3222x INHIBITS α-SYNUCLEIN AGGREGATION 791
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026
(3-nitrophenyl)-2-oxo-4-(trifluoromethyl)-1H-pyridine-
3-carbonitrile) and Buntanetap were obtained from
commercial sources (MedChemExpress, USA).
Cell cultures. SH-SY5Y cells were cultured in
DMEM/F12 medium (PanEco, Russia) supplemented
with 10% fetal bovine serum (HyClone, USA) and
2 mM L-glutamine (PanEco) at 37°C with 5% CO
2
.
Cell viability analysis. Viability of SH-SY5Y cells
was analyzed using an MTS assay kit (Abcam, UK)
according to the manufacturer’s protocol. Cells were
seeded in 96-well plates at a density of 1×10
4
cells
in 100 µL of medium. After 24 h, the test compound,
dissolved in dimethyl sulfoxide (DMSO) and diluted
in 100 µL of medium was added to the experimental
wells. Control wells received DMSO at the same final
concentration as in the experimental samples. Cells
were incubated for another 24 h. Next, 20 µL of an
MTS reagent was added to each well, and the plates
were incubated for additional 1-2 h. Optical density
was measured at 490 nm using a Cytation 3 micro-
plate reader (BioTek, USA). IC50 values were calcu-
lated based on dose-response curves using GraphPad
Prism 8 software (GraphPad Software Inc., USA). Cell
viability in control wells was taken as 100%.
Transfection of cell cultures. A genetic con-
struct encoding the mutant α-synuclein (α-Syn A53T)
based on the pcDNA3.1 vector was kindly provided by
Professor V. L. Buchman. Plasmid DNA was isolated
using a Plasmid Miniprep Color kit (Evrogen, Russia).
Sequence of the insert in the plasmid was confirmed
by Sanger sequencing. Before transfection, SH-SY5Y
cells were grown in 24-well plates on round cover-
slips (12 mm in diameter) pre-coated with poly-L-ly-
sine (PanEco) at a density of 20×10
3
cells per cover-
slip in 500 µL of culture medium. 24 h after seeding,
transfection was performed using a Lipofectamine™
2000 reagent (Thermo Scientific, USA) according to
the manufacturer’s protocol. Test compounds were
added to the cells immediately after transfection at
concentration of 1 µM. Control wells received DMSO
at the same concentration as in the experimental
samples. To induce α-Syn A53T aggregation, 20 h af-
ter transfection, a proteasome inhibitor MG132 (Med-
ChemExpress, USA) dissolved in DMSO was added to
the cells at a concentration of 10 µM followed by a
4-h incubation. For the genetic construct encoding
the mutant form of the transactive response DNA
binding protein 43 kDa (TDP-43) (Δ1-161) [39], trans-
fection and investigation of the activity of chemical
compounds were carried out similarly to the protocol
described above, except that MG132 treatment was
not used, as TDP-43 (Δ1-161) exhibits high propensity
for aggregation on its own.
Immunocytochemical staining (ICC). After
transfection, immunocytochemical staining of cells
was performed. Monoclonal antibodies (clone 4D6)
(ab1903, Abcam) specifically binding to human α-sy-
nuclein were used to detect α-synuclein. To analyze
the effect of the test compound on apoptotic cell
death, staining was performed using antibodies to
the activated form of caspase 3, CC3 (AB3623, Sigma-
Aldrich, USA). Cells were washed with a 1× PBS
(Phosphate Buffered Saline) and fixed with a cold
4% paraformaldehyde solution for 15 min. They
were next treated with cold methanol for 5 min. Cells
were incubated in a blocking buffer consisting of
1× PBS-Tween-20 supplemented with 5% goat serum
for 60 min. This was followed by incubation with
primary antibodies (1 : 1000 dilution) in blocking
solution for 60 min. This was followed by incubation
with fluorescently labeled secondary antibodies Goat
anti-Rabbit IgG Alexa Fluor™ 568 (1 : 1000, A-11011,
Thermo Fisher Scientific, USA) and Goat anti-Mouse
IgG Alexa Fluor™ 488 (1 : 1000, A-11029, Thermo
Fisher Scientific) for 90 min. Cell nuclei were stained
with 0.5 µM DAPI (4′,6-diamidino-2-phenylindole,
Sigma-Aldrich) for 3 min. Coverslips were mounted
using an Immu-Mount medium (Thermo Scientific).
In the case of transfection with the plasmid en-
coding TDP-43 (Δ1-161), only cell fixation and DAPI
staining were performed, as the construct contained
the TDP-43 (Δ1-161) protein fused with green fluores-
cent protein (GFP) as a marker.
Image acquisition and analysis. Fluorescence
microscopy was performed using a Cytation 3 instru-
ment (BioTek, USA) and Gen5 3.08 software (BioTek).
Scanning area was at least 2000×2000 µm. Total num-
ber of cells was counted automatically based on the
signal from the DAPI-stained nuclei. Average number
of cells in the analyzed area was 10,000 per 10 mm
2
and did not differ between the experimental groups.
Confocal images were obtained using a Carl Zeiss
LSM880 microscope (Carl Zeiss, Germany) and ana-
lyzed with the ZEN2.3 software (Carl Zeiss). Cells with
α-synuclein aggregates were counted manually by a
researcher blinded to the sample group. For morpho-
logical analysis of aggregation types, ten non-overlap-
ping fields of view containing clusters of cells with
aggregates were selected for each slide, and counting
was performed. Analysis of micrographs of cultures
after TDP-43(Δ1-161) transfection was performed us-
ing a Carl Zeiss Axio Observer 3 fluorescence micro-
scope equipped with an Axiocam 712 mono camera
and ZEN 2.3 software (Carl Zeiss) and ImageJ with
the Fiji package (Wayne Rasband, USA). Within a sin-
gle experiment, 3-6 independent cell cultures (slides)
were analyzed for each group. The experiment was
reproduced at least twice by independent researchers
for all compounds.
Statistical analysis. Statistical analysis of the
obtained data was performed using the Graph-
Pad Prism 8 software (GraphPad Software Inc.).
BURAK et al.792
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026
One-way ANOVA was used for comparing more than
two groups. The Shapiro–Wilk test and Levene’s test
confirmed normal distribution and homogeneity of
variances, respectively. The Mann–Whitney U test was
used for comparing two groups with non-normal data
distribution. In all cases, results are presented as a
mean ± standard error of the mean. Critical level of
statistical significance was set at p < 0.05. Details of
the statistical analysis for each experiment are pro-
vided in the figure legends.
RESULTS
To select a safe concentration of EC3222x, its tox-
ic effect on SH-SY5Y cells was evaluated in the range
of micromolar concentrations (from 1.6 to 100 µM)
using the MTS assay (Fig. 1). The obtained IC50 value
was 62.34 µM. For further evaluation of anti-aggrega-
tion properties of the compound, a concentration of
1 µM was used, which is more than ten times lower
than the IC50 value. Thus, during the experiments,
EC3222x did not affect viability of SH-SY5Y cells, and
the observed cell death was due to toxicity of α-sy-
nuclein aggregates.
To study anti-aggregation activity of EC3222x,
an in vitro model of mutant α-synuclein A53T ag-
gregation in the SH-SY5Y neuroblastoma cells was
used [40]. Transient transfection of the cell cultures
with the α-SynA53T-pcDNA3.1 genetic construct was
performed, followed by immunocytochemical staining
using antibodies against α-synuclein. To further stim-
ulate aggregate formation, the proteasome inhibitor
MG132 was added to the cells [41]. It is known that
MG132 promotes accumulation of the mutant forms of
α-synuclein and their aggregation [41, 42]. This model
reproduces main molecular pathological events char-
acteristic of synucleinopathies – in some cells, there
is a homogeneous distribution of the protein, while in
others, protein aggregates of several types (granular,
fibrillar, aggresomes) are found, which exert cytotoxic
effects and stimulate apoptotic cell death [40].
Before testing the effects of the compound
EC3222x, we conducted a series of experiments to
validate the model using substances with proven an-
ti-aggregation effects against α-synuclein. SynuClean-D
(SC-D) [20, 21] and Buntanetap (Bun) [22-25] were
used as known aggregation blockers. Immediately
after transfection, SC-D and Bun were added to the
cells at concentration of 1 µM. After 20 h, aggregation
of α-Syn A53T was stimulated in the experimental
and control cultures by adding 10 µM MG132 for 4 h.
The cells were next fixed and immunocytochemical-
ly stained. The number of the cells containing α-Syn
A53T protein aggregates was counted after MG132 ad-
dition. A significant reduction in the number of the
cells with aggregates was observed under the action
of both compounds (Fig. 2), confirming validity of
the model for evaluation of the effects of potential
anti-aggregation substances against α-synuclein.
Next, using the same experimental scheme, we
evaluated the effect of EC3222x on the α-Syn A53T
aggregation. EC3222x reduced the number of cells
containing aggregates (Fig. 2). The observed effect
of EC3222x was comparable to that of the reference
compounds SC-D and Bun. Confocal microscopy was
used for a more detailed analysis of α-synuclein ag-
gregation in the cells, specifically counting the cells
with different types of protein distribution: (i) ho-
mogeneous distribution throughout the cytoplasm;
(ii) granular aggregates – numerous small aggregates
scattered in the cytoplasm; (iii) fibrillar aggregates –
chain-like and ring-like perinuclear strands; (iv) ag-
gresomes – large cytoplasmic inclusions of a rounded
shape with a halo. EC3222x did not significantly affect
the number of cells containing granular aggregates
Fig. 1. Compound EC3222x. a) Structure of EC3222x. b) Viability of SH-SY5Y cells 48 h after treatment with EC3222x in
the range of micromolar concentrations, measured using the MTS assay. One-way ANOVA, F
7, 15
= 50.69; Dunnett’s test for
multiple comparisons, **** p < 0.0001.
EC3222x INHIBITS α-SYNUCLEIN AGGREGATION 793
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026
Fig. 2. Effect of compounds Bun, SC-D, and EC3222x on α-Syn A53T aggregation. a) Representative micrographs (fluorescence
microscopy) of SH-SY5Y cells after transfection with the α-Syn A53T construct followed by treatment with 10 µM MG132
only (MG132), 10 µM MG132 and 1 µM Bun (MG132 + Bun), 10 µM MG132 and 1 µM SC-D (MG132 + SC-D), 10 µM MG132 and
1 µM EC3222x (MG132 + EC3222x) for 4 h. Staining was performed using antibodies against α-synuclein protein (green), and
cell nuclei were stained with DAPI (blue). Scale bar 100 µm. b) Number of cells with aggregates in the analyzed cultures.
One-way ANOVA, F
3, 15
= 41.48; Dunnett’s test for multiple comparisons, **** p < 0.0001.
but reduced the number of cells with homogeneous
protein distribution, fibrillar aggregates, and large ag-
gresomes (Fig. 3). Thus, EC3222x influenced almost all
stages of aggregation, suppressing formation of the
large insoluble aggregates and reducing accumulation
of the initial monomeric protein forms.
Protein aggregates, including those of α-synucle-
in, exert cytotoxic effects, cause cellular stress, and
ultimately lead to cell death [43-45]. To assess the ef-
fect of EC3222x on apoptotic cell death induced by
the α-Syn A53T aggregate formation, we counted the
cells expressing activated form of caspase 3 (CC3) af-
ter ICC. Treatment of the cells with EC3222x did not
affect the extent of apoptotic death in the cultures
(Fig. 4), at least within the selected time frame, i.e.,
24 h after onset of the mutant protein expression and
4 h after the start of proteasome inhibition.
To determine specificity of the EC3222x effects
in reducing α-synuclein accumulation, we examined
its effect on aggregation of another aggregation-prone
protein, TDP-43. TDP-43 (transactive response DNA
binding protein 43 kDa) is a nuclear DNA/RNA-bind-
ing protein with a mass of 43 kDa, expressed in all
cells and playing an important role in RNA metabo-
lism [46, 47]. The TDP-43 aggregation is a key event
in pathogenesis of several NDDs, including amyo-
trophic lateral sclerosis and frontotemporal demen-
tia [48]. To model TDP-43 aggregation in cells, a ge-
netic construct encoding a mutant truncated form
of the TDP-43 (Δ1-161) protein fused with GFP as a
marker was used. Such C-terminal truncated frag-
ments of the protein are found in the aggregates
in NDDs [49]. After transfection with this plasmid
construct, the SH-SY5Y cells developed pronounced
accumulation of TDP-43 (Δ1-161) aggregates within
the first 24 h. Addition of EC3222x immediately after
transfection did not affect accumulation of TDP-43
(Δ1-161) in the cells (Fig. 5).
DISCUSSION
Finding effective treatments for NDDs, includ-
ing synucleinopathies, remains an important and
unresolved medical challenge [11]. There is a need
to develop therapies targeting the key pathogenet-
ic mechanisms underlying disease development, in-
cluding protein aggregation processes [12, 13]. In our
study, we investigated a new compound, EC3222x,
and presented results demonstrating its specific abil-
ity to block accumulation of the pathogenic form of
the α-synuclein A53T protein. EC3222x is a promising
drug candidate with inhibitory activity against α-sy-
nuclein aggregation, which could be used to treat
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BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026
Fig. 3. Effect of compound EC3222x on different types of α-synuclein aggregates. a) Representative micrographs (confocal flu-
orescence microscopy) of cells with different types of α-synuclein accumulation: 1– homogeneous distribution, 2– granular
aggregates, 3– fibrillar aggregates, 4– large aggresomes (insets show individual cells at higher magnification). Staining was
performed using antibodies against α-synuclein protein (green), and cell nuclei were stained with DAPI (blue). Scale bar:
50 µm. b) Number of cells with different types of aggregates in the analyzed cultures after treatment with 10 µM MG132 only
(MG132) or 10 µM MG132 and 1 µM EC3222x for 4 h (MG132 + EC3222x). Two-way ANOVA, for the factor “type of aggregates”
F
3, 28
= 8.401, for the factor “compound EC3222x” F
1, 28
= 17.91, Fisher’s test for multiple comparisons, * p < 0.05, ** p < 0.01.
Fig. 4. Effect of compound EC3222x on apoptotic cell death. a) Representative micrographs (fluorescence microscopy) of
SH-SY5Y cells after transfection with the α-Syn A53T construct followed by treatment with 10 µM MG132 only (MG132)
or 10 µM MG132 and 1 µM EC3222x (MG132 + EC3222x) for 4 h. Staining was performed using antibodies against α-sy-
nuclein protein (green) and antibodies against the activated form of caspase 3 (CC3), and cell nuclei were stained with
DAPI (blue). Scale bar: 100 µm. b) Number of cells expressing the apoptosis marker (activated form of caspase 3, CC3).
Mann–Whitney U test.
EC3222x INHIBITS α-SYNUCLEIN AGGREGATION 795
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Fig. 5. Effect of compound EC3222x on TDP-43 (Δ1-161) aggregation. a) Representative micrograph (fluorescence microscopy)
of SH-SY5Y cells after transfection with the TDP-43 (Δ1-161) construct (green signal from GFP) after treatment with 1 µM
EC3222x or without it. Cell nuclei were stained with DAPI (blue). Scale bar: 50 µm. b) Number of cells with aggregates in
the analyzed cultures. Mann–Whitney U test.
corresponding proteinopathies. The initial design of
EC3222x is based on combining two pharmacoph-
ores – adamantane and carboline fragments – within
a single molecule, each of which individually exhib-
its neuroprotective and anti-Parkinsonian properties
[28-33, 36, 37]. This combination has created a mol-
ecule with new biological properties useful for com-
bating NDDs [38].
EC3222x exhibited low toxicity in the SH-SY5Y
cells and, at non-toxic concentration (1 µM), signifi-
cantly reduced the number of cells with aggregates
of the mutant α-synuclein A53T protein. Importantly,
this effect was comparable to that of the compounds
with known anti-aggregation properties, SynuClean-D
and Buntanetap, highlighting pronounced ability of
EC3222x to block accumulation of the mutant α-sy-
nuclein. In addition to assessing total number of the
cells containing aggregates, we analyzed the effect
of EC3222x on different types of α-Syn A53T aggre-
gates. No significant differences were found in the
number of cells with small granular aggregates. It is
worth noting that their proportion among total ag-
gregates is relatively low, possibly due to their short
lifetime and tendency to rapidly merge into larger
structures. At the same time, EC3222x significantly
reduced the number of cells with fibrillar aggregates
and large aggresomes. This suggests that EC3222x ef-
fectively slows down accumulation and maturation
of oligomers transitioning into the large insoluble
aggregates. Given that large aggresomes are particu-
larly resistant to cellular clearance mechanisms and
significantly contribute to cytotoxicity [50], inhibiting
these late stages of aggregation is of great therapeu-
tic importance. The action of EC3222x may be due
to reduction in the level of monomeric α-synuclein,
blockage of interaction of the preformed oligomers
and protofibrils, and/or acceleration of the removal
of intermediate aggregated protein forms.
It was also found that EC3222x did not alter the
level of apoptotic cell death in the context of α-Syn
A53T aggregate formation, despite the significant re-
duction of the number of large aggregates. On the
one hand, this may be explained by the fact that the
small aggregates exhibit greater cytotoxicity than the
large ones [51], and since EC3222x did not reduce the
number of granular aggregates, cell death in the cul-
tures remained unchanged. On the other hand, cell
death may occur through the caspase 3-independent
mechanisms, which were not assessed in our study.
These results highlight complexity of the impact of
α-synuclein aggregation on cell fate. Further studies
are needed to elucidate the mechanisms of EC3222x
neuroprotective activity, including a more detailed
characterization of cell death.
An important issue is specificity of the inhib-
itory action of the tested compounds on protein
aggregation. During characterization of each such
compound, it is necessary to determine whether its
anti-aggregation properties are universal and could
be manifested against different proteins, for example,
BURAK et al.796
BIOCHEMISTRY (MOSCOW) Vol. 91 No. 5 2026
by binding the related structural motifs, or whether
they are specific to a particular protein. For potential
therapeutic development, it is essential to determine
whether a compound can selectively inhibit the ag-
gregation of specific proteins. According to the re-
sults of this study, EC3222x exhibits some specificity
for α-synuclein and does not inhibit aggregation of
another protein, TDP-43 (Δ1-161), which is involved
in pathogenesis of amyotrophic lateral sclerosis and
frontotemporal dementia [48]. Further studies involv-
ing models of aggregation of other proteins will show
how selectively EC3222x acts against α-synuclein.
CONCLUSION
In this study, we described the compound
EC3222x, which can block aggregation of the mu-
tant α-synuclein. Its efficacy, comparable to that of
the known anti-aggregation substances, specificity of
action, and inhibition of formation of the final ag-
gregation products make EC3222x a promising drug
candidate for further development. Future research
should focus on elucidating the mechanism of action,
precise molecular targets, and testing efficacy of this
compound in in vivo models of synucleinopathies.
Abbreviations
AD Alzheimer’s disease
Bun buntanetap
DAPI 4′,6-diamidino-2-phenylindole
DMSO dimethyl sulfoxide
GFP green fluorescent protein
NMDA N-methyl-D-aspartate
PD Parkinson’s disease
SC-D SynuClean-D
TDP-43 transactive response DNA binding
protein 43 kDa
Contributions
M. V. Burak and N. E. Pukaeva– planning and conduct-
ing experiments, manuscript preparation; O. A. Kukhar-
skaya – processing and analysis of the primary data,
manuscript editing; V. S. Kryshkova, M. R. Nazdracheva,
S. A. Pukhov, and K. A. Rachenkov – planning and con-
ducting experiments; A. V. Stavrovskaya, V. P. Fisenko,
and M. S. Kukharsky – analysis and interpretation of
the results, manuscript preparation; S. O. Bachurin –
research planning, data analysis and interpretation,
manuscript preparation and editing.
Funding
This work was financially supported by the program
of the Ministry of Science and Higher Education of
the Russian Federation for implementation of major
scientific projects in the priority areas of scientific
and technological development (project no. 075-15-
2024-638).
Ethics approval and consent to participate
This work does not contain any studies involving hu-
man and animal subjects.
Conflict of interest
The authors of this work declare that they have no
conflicts of interest.
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