Discovery of a necroptosis inhibitor improving dopaminergic neuronal loss after mptp exposure in mice

Article abstract of DOI:10.3390/ijms22105289

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, mainly characterized by motor deficits correlated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SN). Necroptosis is a caspase-independent f

Full text of DOI:10.3390/ijms22105289

International Journal of
Molecular Sciences
Article
Discovery of a Necroptosis Inhibitor Improving Dopaminergic
Neuronal Loss after MPTP Exposure in Mice
Sara R. Oliveira , Pedro A. Dionísio, Maria M. Gaspar , Maria B. T. Ferreira, Catarina A. B. Rodrigues,
Rita G. Pereira, Mónica S. Estevão, Maria J. Perry, Rui Moreira , Carlos A. M. Afonso , Joana D. Amaral
and Cecília M. P. Rodrigues *
Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa,
1649-003 Lisbon, Portugal; sararoliveira@ff.ulisboa.pt (S.R.O.); pedelandionisio@gmail.com (P.A.D.);
mgaspar@ff.ulisboa.pt (M.M.G.); mariabtferreira@gmail.com (M.B.T.F.); cataxana@gmail.com (C.A.B.R.);
ragpereira@gmail.com (R.G.P.); monica.estevao@gmail.com (M.S.E.); mjprocha@ff.ulisboa.pt (M.J.P.);
rmoreira@ff.ulisboa.pt (R.M.); carlosafonso@ff.ulisboa.pt (C.A.M.A.); jamaral@ff.ulisboa.pt (J.D.A.)
* Correspondence: cmprodrigues@ff.ulisboa.pt; Tel.: +351-217946490
Abstract: Parkinson’s disease (PD) is the second most common neurodegenerative disorder, mainly
characterized by motor deficits correlated with progressive dopaminergic neuronal loss in the
substantia nigra pars compacta (SN). Necroptosis is a caspase-independent form of regulated cell
death mediated by the concerted action of receptor-interacting protein 3 (RIP3) and the pseudokinase
mixed lineage domain-like protein (MLKL). It is also usually dependent on RIP1 kinase activity,
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
influenced by further cellular clues. Importantly, necroptosis appears to be strongly linked to several
ꢀꢁꢂꢃꢄꢅꢆ
neurodegenerative diseases, including PD. Here, we aimed at identifying novel chemical inhibitors of
necroptosis in a PD-mimicking model, by conducting a two-step screening. Firstly, we phenotypically
Citation: Oliveira, S.R.; Dionísio,
P.A.; Gaspar, M.M.; Ferreira, M.B.T.;
screened a library of 31 small molecules using a cellular model of necroptosis and, thereafter, the hit
Rodrigues, C.A.B.; Pereira, R.G.;
compound effect was validated in vivo in a sub-acute 1-methyl-1-4-phenyl-1,2,3,6-tetrahydropyridine
Estevão, M.S.; Perry, M.J.; Moreira, R.;
hydrochloride (MPTP) PD-related mouse model. From the initial compounds, we identified one
Afonso, C.A.M.; et al. Discovery of a
hit—Oxa12—that strongly inhibited necroptosis induced by the pan-caspase inhibitor zVAD-fmk in
the BV2 murine microglia cell line. More importantly, mice exposed to MPTP and further treated
with Oxa12 showed protection against MPTP-induced dopaminergic neuronal loss in the SN and
striatum. In conclusion, we identified Oxa12 as a hit compound that represents a new chemotype
to tackle necroptosis. Oxa12 displays in vivo effects, making this compound a drug candidate for
further optimization to attenuate PD pathogenesis.
Necroptosis Inhibitor Improving
Dopaminergic Neuronal Loss after
MPTP Exposure in Mice. Int. J. Mol.
Sci. 2021, 22, 5289. https://doi.org/
10.3390/ijms22105289
Academic Editor: Anne Vejux
Keywords: MPTP; necroptosis; neurodegeneration; Parkinson’s disease; small molecules
Received: 16 April 2021
Accepted: 14 May 2021
Published: 18 May 2021
1. Introduction
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Parkinson’s disease (PD) is the second most common neurodegenerative disorder
worldwide. PD is pathologically defined by the progressive dysfunction of the nigrostriatal
pathway, which culminates in the loss of dopaminergic neurons in the substantia nigra
pars compacta (SN) and depletion of dopaminergic enervation in the striatum [1,2]. Of
note, degeneration of dopaminergic neurons in the SN precedes the first motor deficits
afflicting PD patients [2].
The pathological mechanisms causing PD are thought to stimulate a cascade of events
that activate regulated cell death (RCD) pathways, which are responsible for neuronal
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This article is an open access article
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Attribution (CC BY) license (https://
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4.0/).
death [
plays crucial pathogenic roles in several human diseases, including neurodegenerative
diseases, such as PD, while holding high potential for clinical targeting [ ]. Necroptosis
3,4]. Recent evidence has shown that necroptosis, a type of regulated necrosis,
5
is a caspase-independent type of RCD commonly executed after RIP1 and RIP3 kinase
activation. Typically, this type of cell death is initiated following cell death transmembrane
receptor stimulation, with tumor necrosis factor (TNF) receptor 1 (TNFR1) being the most
Int. J. Mol. Sci. 2021, 22, 5289. https://doi.org/10.3390/ijms22105289
https://www.mdpi.com/journal/ijms

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well-studied example [
6,
7]. In conditions where caspase-8 activation is genetically or phar-
macologically prevented, RIP1 and RIP3 are not cleaved and accumulate in the so-called
necrosome complex [ ]. Then, activated RIP3 recruits and phosphorylates pseudokinase
mixed lineage domain-like protein (MLKL), which further translocates to the plasma
membrane, inducing membrane disruption and necroptosis execution [ ]. Importantly,
8
5
necroptosis has already been associated with neuronal death induced by the neurotoxin
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a PD-mimicking neurotoxin, in both
in vivo and in vitro rodent models, as well as in PD human samples [9,10].
Importantly, the first necroptosis inhibitors were identified back in 2005 through a
phenotypic screening for chemical inhibitors of necroptosis induced by TNF and zVAD-fmk
in human monocytic U937 cells. This led to the identification of necrostatin-1 (Nec-1) and
its optimized derivative necrostatin-1 stable (Nec-1s), later confirmed to be RIP1 kinase
inhibitors [11–13]. Additional studies regarding Nec-1 properties pointed to off-target
activity and limited metabolic stability in mice, while Nec-1s presented higher activity as a
necroptosis inhibitor, along with no nonspecific cytotoxicity and reasonable pharmacoki-
netic properties. However, Nec-1s still presents a short in vivo half-life [11,14–17]. Of note,
Nec-1s identification proved that RIP1 inhibition is beneficial in several diseases [
Moreover, molecules targeting other components of necroptotic signaling pathway, such
as RIP3 or MLKL, have also been proposed [19 20]. However, none of the compounds
9,10,13,18].
,
discovered so far reached the expectation of clinical application, which has led researchers
to unceasingly screen new drugs with better selectivity and potency.
In this study, we phenotypically screened a library of 31 new molecules to discover
novel inhibitors of necroptotic cell death with structural novelty. Although target-based
drug discovery has been the dominant approach to drug discovery in the past decades,
there has been a recent reawakening interest in phenotypic drug discovery approaches,
based on their potential to address the complexity of only partially understood diseases
and their promise of delivering first-in-class drugs, in parallel with major advances in
cell-based phenotypic screening tools. In addition, prioritized hits from the primary screen
were further evaluated for in vivo proof-of-concept using an MPTP PD-mimicking mouse
model. We identified a potential hit, Oxa12, that significantly inhibited zVAD-fmk-induced
necroptotic cell death in the BV2 microglial cell line, with a half-maximal effective concen-
tration (EC50) of ~1 µM. Importantly, Oxa12 showed to protect dopaminergic neuronal
cells from death induced by MPTP in vivo, in the SN and striatum, which highlights the
potential benefits of discovering and optimizing new molecules with mechanisms of action
that affect disease-relevant pathways, such as necroptosis.
2. Results
2.1. Phenotypic Screening for Hit Selection
To identify novel necroptosis inhibitors, we performed a cell-based phenotypic screen-
ing assay to select hit compounds that strongly inhibit necroptotic cell death (Figure 1A).
Previous studies have demonstrated that the pan-caspase inhibitor zVAD-fmk induces
necroptosis in different cellular models, including in the L929 fibrosarcoma and in the BV2
murine microglial cell lines, by a mechanism that most likely depends on TNF autocrine
secretion [12,21,22]. Here, we used BV2 cells incubated with zVAD-fmk as an in vitro
model of necroptosis and screened a total of 31 small compounds potentially inhibitors of
this type of cell death. These compounds are part of a larger in-house library containing
mainly the heterocyclic core of 4-methylene-2-aryloxazol-5(4H)-one and are represented
in Figure 2. The remaining compounds have been tested in previous papers [12
,21,22].
Cells were incubated with 25 M zVAD-fmk alone or in combination with 30
µ
µM test
compounds for 24 h. BV2 cells treated with DMSO were used for data normalization. BV2
cells co-incubated with zVAD-fmk and Nec-1, a well-known RIP1 kinase inhibitor, were
used as positive control of necroptosis inhibition (Figure 1B). As expected, treatment with
Nec-1 almost completely rescued zVAD-fmk-induced decrease in cell viability (p < 0.001
)
(Figure 1B), as assessed by MTS metabolism. More importantly, we identified one hit

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compound—Oxa12—that significantly protected cells from zVAD-fmk-mediated necropto-
sis by ~70% (p < 0.01) (Figure 1B). Importantly, RIP1 kinase activity inhibitory potential
of Oxa12 was determined using radiometric binding-based assays. The results show that
Oxa12 at 5 µM inhibits RIP1 activity by ~15%, which confirms RIP1 as a possible target. The
anti-necroptotic effects, however, may not be solely dependent on this target. These results
are in line with our previous work, where we showed the sequestration of key necroptosis
mediators, RIP1, RIP3, and p-MLKL, in the insoluble fraction in zVAD-fmk-treated BV2
cells, while Oxa12 abolished necrosome assembly and MLKL phosphorylation [22]. More-
over, Oxa12 strongly rescued zVAD-fmk-induced cell death, as observed by microscopy
analysis of cell morphology [22]. Furthermore, our previous in silico molecular docking
calculations for Oxa12 inside the RIP1 kinase domain demonstrated that Oxa12 is occupy-
ing a region similar to the co-crystallized inhibitor, suggesting a similar interaction pattern
and indicating that Oxa12 most likely targets RIP1 to some extent [22].
Figure 1. A cell-based phenotypic screening identifies Oxa12 as a necroptosis inhibitor. (
step screening workflow. ( ) Determination of the ability of the compound to inhibit necroptosis. Cell metabolic activity is
depicted as a percentage of the control (DMSO = 0; Nec-1 at 30 M) for compounds at 30 M tested in BV2 murine microglia
cells exposed to 25 M zVAD-fmk for 24 h. ( ) Half-maximal effective concentration (EC50) determination in BV2 murine
microglia cells in a dose-response concentration (0.1 to 50 M Oxa12) plus 25 M zVAD-fmk for 24 h. ( ) Half-maximal
A) Schematic overview of the two-
B
µ
µ
µ
C
µ
µ
D
effective concentration (EC50) determination in BV2 murine microglia cells in a dose-response concentration (0.01 to 50 µM
Nec-1) plus 25 µM zVAD-fmk for 24 h.

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Figure 2. Structures of the synthesized small-molecule library compounds based on the 4-methylene-2-aryloxazol-5(4H)-one
core (oxazolones). Group A includes oxazolones containing substituted aryl and aromatic heterocyclic attached to the
4-methylene position. Group B consists of extended oxazolones containing terminal fused heterocycles. Group C includes
a
additional compounds without the oxazolone core or the 4-methylene side chain. Synthesized compounds evaluated in a
b
previous paper [22]. Compounds not evaluated due to insoluble properties at the tested concentration.
To further characterize Oxa12 activity, we determined the EC50, a pharmacologic
parameter commonly used as a measure of compound potency, which here indicates 50%
of compound maximal potency to inhibit necroptosis, by performing a dose-response study.
BV2 cells were incubated with zVAD-fmk along with Oxa12 at a range of concentrations
(
0.1–50 µM) for 24 h and the EC50 was calculated. Our results showed that Oxa12 pre-
sented an EC50 value of 0.989 M, comparable with 0.124 M of Nec-1, thus indicating its
effectiveness as a necroptosis inhibitor (Figure 1C,D).
µ
µ
We next found that Oxa12 met stringent criteria on activity, chemical tractability and
structural novelty, including a low chemical similarity with Nec-1 and its analogues, as
measured by Tanimoto’s index. Moreover, we analyzed whether Oxa12 was included
in the central nervous system (CNS) drug property space, using a CNS multiparameter
optimization (MPO) approach, a prospective design tool and a widely utilized predictor
for ADME and safety properties suitable for CNS targeting [23]. CNS MPO utilizes a set
of six physicochemical descriptors to calculate a score, between 0 and 6. A higher score
represents the optimal chemical space possessing alignment of key drug properties for
CNS therapeutic agents. Generally, a CNS MPO score
≥
4 is desired, although there are

Int. J. Mol. Sci. 2021, 22, 5289
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drugs active on the CNS with lower score, with a range of 3–4 being still acceptable [24].
Here, we observed that Oxa12 has a value of 3.6. While being lower than the CNS MPO
score of 5 calculated for Nec-1, the value for Oxa12 is similar to that determined for several
well-known CNS drugs, suggesting good brain exposure (Table 1).
Table 1. Central nervous system MPO calculations for Oxa12 and Nec-1.
Oxa12
Individual Score
Nec-1
Individual Score
Score CNS
MPO
Score CNS
MPO
Physiochemical Descriptor
Physiochemical Descriptor
MW
clogP
clogD7.4
TPSA
HBD
391.43
5.29
5.69
67.34
1
0.8
0
MW
clogP
clogD7.4
TPSA
HBD
259.33
1.66
1.66
48.13
2
1.0
1.0
1.0
1.0
0.5
1.0
0
3.6
5.5
1.0
0.8
1.0
pKa
5.34
pKa
0
MW, molecular weight; clogP, calculated logP (n-octanol/buffer distribution coefficient) using Molinspiration desktop property calculator
(https://www.molinspiration.com/cgi-bin/properties, accessed on 15 April 2021); clogD7.4, distribution coefficient at pH 7.4; TPSA,
topological polar surface area; HDB, number of hydrogen bond donor atoms; pKa, acidity constant of ionizable function.
Finally, we also determined the extent of Oxa12 metabolism in mouse liver microsomes.
Using our medium-throughput drug metabolism platform, we observed that 20% of Oxa12
was detected by HPLC after incubation for 30 min in mouse liver microsomes, which
corresponds to an estimated half-life of 13 min, a liability that negatively impacts the
systemic and brain exposure to Oxa-12. These results allowed us to calculate a clearance
Clint in vitro of 0.99 mL/min/mg protein and a predicted hepatic extraction ratio of 0.76.
Of note, although the original Nec-1 has a half-life of < 5 min, chemical optimization of
this compound has led to commonly used derivatives with a half-life of about 1 h in mouse
microsomal assays [25]. Thus, a combined analysis of the CNS MPO score and metabolic
data confirms that Oxa12 is a novel addition to the chemical toolbox of CNS-targeting
anti-necroptotic compounds.
2.2. In Vivo Efficacy of Oxa12 in the Sub-Acute MPTP Mouse Model
To determine if our identified hit compound was effective in vivo, we used a sub-
acute MPTP mouse model. MPTP is the most widely used neurotoxin to mimic PD-related
nigrostriatal degeneration, since it can be easily administered systemically, and human
exposure to MPTP has been associated with severe parkinsonism, which reinforces the
relevance of MPTP animal models [26,27]. Mechanistically, MPTP can rapidly cross the
blood–brain barrier and, once in the brain, it is mostly oxidized to an intermediate, which
then diffuses to the extracellular space and converts to the active toxic metabolite, 1-methyl-
4-phenylpyridinium (MPP+) [28 29]. Then, dopaminergic neurons selectively uptake MPP+.
,
In mitochondria, MPP+ inhibits the complex I of the respiratory chain, leading to decreased
ATP generation along with the production of reactive oxygen species (ROS), and ultimately
cell death [29,30]. In this regard, several studies have already demonstrated that necroptosis
is involved in neuronal death induced by MPTP and that Nec-1/Nec-1s administration is
neuroprotective in multiple MPTP exposure regimens [9,10].
Here, we used a sub-acute MPTP regimen consisting of a single dose of MPTP
(40 mg/kg), or vehicle, intraperitoneally injected in 13-week-old mice. Then, 10 mg/kg of
Oxa12 or Nec-1s was administered 1 h after MPTP and then once every day for 30 days. As
expected, we observed a significant reduction of approximately ~50% in TH-positive stain-
ing, a marker of dopaminergic neurons, in the SN and in the striatum of MPTP-exposed
animals (Figure 3A,B), while no differences were observed in mice injected with Oxa12 or
Nec-1s alone (Figure 3A,B).

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Figure 3. Oxa12 protects from MPTP-driven dopaminergic neuronal loss. Representative images of TH-positive immunos-
taining from control- and MPTP-injected mice treated with vehicle, Nec-1s or Oxa12 in the SN (A) and the striatum (B), and
respective quantification. Scale bar, 100 µm. * p < 0.05 vs. control mice.
Importantly, treatment with Oxa12 or Nec-1s partially protected cells from MPTP-
induced dopaminergic cell death, in the SN (Oxa12, p = 0.05; Nec-1s, p = 0.12) and striatum
(Oxa12, p = 0.06; Nec-1s, p = 0.08), as observed by the rescue of TH-positive staining in
comparison with MPTP-treated animals (Figure 3A,B).
In accordance, exposure to MPTP significantly reduced TH protein levels in the SN,
while Oxa12 and Nec-1s treatment alone did not alter TH levels (Figure 4). Notably, Oxa12
significantly rescued TH protein levels by ~30% (p < 0.05), while Nec-1s showed a ~10%
difference (Figure 4). The challenging dissection of SN-enriched midbrain sections, which
include other dopaminergic structures, may account for the less significant rescue of TH
in the SN by western blot as compared with immunofluorescence. Our results show that
Oxa12 reveals a propensity to protect neuronal cells from MPTP-induced cell loss, with
the potential to be explored as a new chemotype to tackle necroptosis. Nevertheless,
careful examination of target engagement, including RIP1 and MLKL sequestration in
insoluble fractions, a strong indicator of necrosome assembly and, therefore, of necroptosis
commitment [28], deserves further investigation using appropriate antibodies.

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Figure 4. Oxa12 protects dopaminergic neurons from MPTP-induced cell death. Representative
western blot of TH protein levels from control and MPTP-injected mice untreated or treated with
Nec-1s or Oxa12 in the SN, and the respective densitometric analysis.
β-actin was used as loading
control. Values are expressed as mean SEM of three independent experiments. * p < 0.05 vs. control
±
mice; § p < 0.05 vs. MPTP mice. TH, tyrosine hydroxylase.
Finally, MPTP mice models normally develop an inflammatory response that initiates
in the striatum, with astrogliosis developing later than microgliosis and being sustained
for a longer period of time [29,30]. Concordantly, we observed an increase in GFAP im-
munostaining and GFAP protein levels in MPTP-treated mice, thus suggesting a prolonged
astrogliosis in these animals (data not shown). However, no significant differences were
observed in mice treated with Oxa12 or Nec-1s.
3. Discussion
In the present study, a two-step screening workflow was performed to identify novel
necroptosis inhibitors from a small-molecule library of 31 compounds. In a first step,
we conducted an in vitro phenotypic screening to identify hit compounds that strongly
inhibited zVAD-fmk-induced necroptotic cell death in the murine BV2 microglial cell line.
In a second step, the ability of our hit molecule to protect against dopaminergic neuronal
cell loss was determined in vivo using the sub-acute MPTP mouse model of PD.
As a strategy for the first step, we initially screened a range of diverse heterocyclic
functionalized compounds collected from different developed synthetic methodologies.
From this preliminary screening, several existing compounds containing the 4-methylene-2-
aryloxazol-5(4H)-one (oxazolones) core were identified as bioactive [31]. Furthermore, we
also synthesized a range of oxazolones containing diverse substituents at the 4-methylene
and 2-aryl positions (Groups A and B) and by replacing the oxazolone core (Group C) as
well as by inclusion of longer phenylmethylene spacer and additional structural tuning
on the attached fused heterocycles (Group B). Importantly, we identified one molecule—
Oxa12—that strongly inhibited necroptosis in the zVAD-fmk-treated BV2 microglia cells,
and this effect was further demonstrated in vivo. Oxa12 tended to protect dopaminer-
gic neuronal cells from MPTP-induced cell death, thus suggesting the potential of this
compound in ameliorating PD pathogenesis.
Since the discovery of Nec-1 as the first necroptosis inhibitor, several other studies
were performed and new inhibitors for RIP1, RIP3 and MLKL were identified. However,
the therapeutic potential of all these inhibitors is restricted by low potency or reduced selec-
tivity. In fact, so far, there is no compound that reached the prospects of clinical application,
which has led to the continuous screening of new molecules with higher selectivity and
potency. Despite its strongness at inhibiting necroptosis, Nec-1 is a far-from-ideal drug due
to its short in vivo half-life of approximately 1 h, as well as its off-target effects, including

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inhibitory binding to indoleamine 2,3-dioxygenase (IDO), a protein involved in inflamma-
tion [32]. Nec-1s presents higher specificity and improved pharmacokinetics properties;
however, it still has poor in vivo half-life [33]. Thus, Nec-1s represents a better choice for
in vivo experiments. Other RIP1 inhibitors, including GSK’481, GSK’963 and GSK’772,
demonstrate similar limitations [34–36]. RIP3 inhibitors, such as GSK’872, have also been
studied; however, while they inhibit necroptosis, they may also promote apoptosis [20].
Thus, considering the limitations of all known RIP1 and RIP3 inhibitors, the development
of novel and potent small compounds able to specifically attenuate necroptosis continues
to be of strong need.
Here, we used the BV2 murine microglia cell line treated with the pan-caspase in-
hibitor, zVAD-fmk, as a model of necroptosis execution. In fact, we and others already
demonstrated that zVAD-fmk strongly induces necroptotic cell death in both L929 fi-
brosarcoma and BV2 murine microglia cells [12,21,22]. Importantly, the pharmacological
relevance of the in vitro phenotypic screening was guaranteed by using a well-established
necroptosis inhibitor. Here, Nec-1, which inhibits RIP1 kinase activity, was used as positive
control throughout the in vitro screening and demonstrated to be a strong necroptosis in-
hibitor. To identify novel necroptosis inhibitors, an in-house library of 31 small compounds
were screened at a concentration of 30 µM, which allowed us to filter new necroptosis
inhibitor scaffolds with potential to be further modified chemically. We identified one
hit—Oxa12—that strongly inhibited necroptosis in the BV2 microglia cells by ~70%. To
further characterize Oxa12 potency in vitro, dose-response curves were performed, where
Oxa12 showed an EC50 value of 0.989 µM, thus reflecting its potential to be tested in vivo.
Throughout the years, MPTP mouse models have been widely used due to their
specific and reproducible neurotoxic effect on the nigrostriatal system, being considered a
convenient model of dopaminergic neurodegeneration to study therapeutic interventions.
However, these models do not fully reproduce the human condition, and behavioral ab-
normalities are still a challenging question [36]. Moreover, previous studies have already
demonstrated the involvement of necroptosis in the acute and sub-chronic MPTP mouse
model of PD, where Nec-1/Nec-1s administration attenuated dopaminergic neurodegener-
ation [9,10]. Here, we used a sub-acute regimen of MPTP exposure, consisting of a single
injection of MPTP (40 mg/kg) and still observed a significant increase in dopaminergic neu-
rodegeneration in the SN and the striatum, while Nec-1s administration showed a tendency
to protect cells from MPTP-induced dopaminergic cell death [27,37,38]. Importantly, Oxa12
alone did not induce neuronal toxicity, but it was able to protect dopaminergic neurons
from MPTP-induced cell death in both SN and striatum. This experimental observation
together with the MPO value suggest that Oxa-12 can cross the BBB, at least to some extent.
Medicinal chemistry optimization of Oxa-12 should tackle issues such as solubility and
be followed by extensive in vivo characterization of a more advanced lead compound,
including by evaluating BBB penetration, half-life and target engagement. The role for
inhibiting necroptosis deserves further exploitation [39].
Overall, these results indicate that Oxa12 has reasonable potency, associated with
an apparent lack of toxicity, which makes this compound fit for additional chemistry
optimization. Therapies using small molecules targeting key components of dopaminer-
gic neuron cell death could evolve as a potential therapeutic approach for ameliorating
PD progression.
4. Materials and Methods
4.1. Cell Culture and Reagents
The BV2 murine microglia cell line (gently provided by Dr. Elsa Rodrigues, Uni-
versity of Lisbon) was cultured in RPMI 1640 medium (GIBCO® Life Technologies, Inc.,
Grand Island, NY, USA), supplemented with 10% heat inactivated fetal bovine serum
(FBS), 1% antibiotic/antimycotic (A/A) solution and 1% GlutaMAXTM (GIBCO). During
the experiments, culture media was substituted by RPMI supplemented with 1% A/A,
1% insulin-transferrin-selenium (RPMI/ITS) and 1 mg/mL bovine serum albumin (BSA;

Int. J. Mol. Sci. 2021, 22, 5289
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GIBCO). Cells were maintained at 37 ◦C in a humidified atmosphere of 5% CO2. Chemi-
cals used were Nec-1 (Sigma-Aldrich, St. Louis, MO, USA), dimethyl sulfoxide (DMSO;
Sigma-Aldrich) and Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) pan-caspase inhibitor
(Enzo Life Sciences, Farmingdale, NY, USA).
4.2. Chemical Synthesis and Analysis
The oxazol-5-(4H)-ones (Oxas) and the precursors aldehydes were synthesized fol-
lowing a general synthetic reported route: a mixture of the corresponding aldehyde
(
30–600 µmol scale, 1 equiv.), hippuric acid or derivatives (1 equiv.), sodium acetate
(1 equiv.) and acetic anhydride (3 equiv.) in a round-bottom flask was heated under
magnetic stirring at 110 ◦C for 2 h [31]. Then, the reaction mixture was cooled to room
temperature and the obtained solid was washed with distilled water (2
×
) and MeOH (4 )
×
and dried under vacuum. If further purification was needed, some of the compounds were
recrystallized in a suitable solvent. The purity and structural identification were performed
by 1H, 13C NMR and mass spectrometry analyses.
4.3. Screening of Necroptosis Inhibitors
BV2 cells were plated in 96-well plates at 7
tosis was induced by adding 25 M zVAD-fmk in RPMI/ITS. Test compounds or Nec-1
(positive control of necroptosis inhibition) were incubated along with zVAD-fmk at a final
×
103 cells/well and after 24 h, necrop-
µ
concentration of 30 µM for 24 h. DMSO was used as vehicle control. Cell viability was
determined based on measurement of MTS metabolism using the CellTiter 96® Aqueous
Non-Radioactive Cell Proliferation (MTS) Assay (Promega, Madison, WI, USA). Differ-
ences in absorbance were measured at 490 nm using GloMax® Multi Detection System
(Sunnyvale, CA, USA).
4.4. EC50 Determination
To quantitatively determine the effective potency in BV2 cells, the compound half
maximal effective concentration or EC50 was determined by a dose-response curve. In brief,
BV2 cells were plated in 96-well plates as described above and treated with increasing
concentrations of compound Oxa12 (from 0.1 to 50 µM) and Nec-1 (from 0.01 to 50 µM)
using DMSO as vehicle control. Following 24 h of incubation, cell viability was determined
based on the measurement of MTS, as previously described.
4.5. RIP1 and RIP3 Kinase Activity Assays
Oxa12 were tested at 5 µM for RIP1 kinase activity by a radiometric-binding assay
using myelin basic protein as substrate (Eurofins, France) as before [39].
4.6. Microsomal Stability Assay
In the microsomal stability assay, an analytical high-performance liquid chromatogra-
phy (HPLC) system was used with the following condition: column, Merck Lichrospher
100 RP18 125 mm 4.6 mm (5
B = 0.1% trifluoroacetic acid in acetonitrile, isocratic; flow rate, 1 mL/min; detection, UV at
400 nm injection, 20 L; column temperature, ambient. The metabolic stability assays were
µm); mobile phase A = 0.1% trifluoroacetic acid in water,
µ
carried out using the cosolvent method, appropriate for assessing the metabolic stability of
compounds poorly soluble in aqueous medium [40]. For the cosolvent method, a 0.5 mM
DMSO stock solution of Oxa12 was prepared. Then, a diluted solution of the compound
was prepared by adding 50
to make a 0.1 mM solution of Oxa12 in 20% DMSO/80% acetonitrile. Cosolvent assay
conditions were: substrate concentration, 1 M; microsomal protein, 0.5 mg/mL; organic
µL of the previous 0.05 mM solution with 200 µL of acetonitrile,
µ
solvents, 0.2% DMSO, 0.8% acetonitrile; incubation time, 30 min; number of assays, dupli-
cates for T0 and T30 min. Time 0 and time 30 batches, after quenching with acetonitrile,
were centrifuged at 11,000
order to quantify compound Oxa12.
×
g for 5 min and the supernatants were analyzed by HPLC, in

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4.7. MPTP Mouse Model
Animal studies were performed according to the animal welfare of the Faculty of
Pharmacy, University of Lisbon, and approved by the competent national authority Direção-
Geral de Alimentação e Veterinária (DAGV) and in accordance with the EU Directive
(2010/63/UE), Portuguese laws (DR 113/2013, 2880/2015 and 260/2016) and all relevant
legislation. To evaluate the neuroprotective effect of our hit compound—Oxa12—in the
sub-acute MPTP mouse model, male 13-week-old C57BL/6N wild-type (wt) mice (Charles
River Laboratories, Wilmington, MA, USA) were injected intraperitoneally (i.p.) with a
unique dose of MPTP-HCl (40 mg/kg; Sigma Aldrich, St Louis, MO, USA), dissolved in
sterile 0.9% saline, or vehicle only (control group). One hour after MPTP administration,
mice were intraperitoneally injected with 10 mg/kg Oxa12 solubilized in 1% DMSO (Sigma-
Aldrich) and 30% 2-hydroxypropyl-beta-cyclodextrin (Sigma-Aldrich) or 10 mg/kg Nec-
1s (Focus Biomolecules, Plymouth Meeting, PA, USA) solubilized in 1% DMSO, 4% 2-
hydroxypropyl-beta-cyclodextrin (Sigma-Aldrich), in PBS [38
injections were administered once every day for 30 days. Oxa12 dosage and regimen of
administration were selected based on published protocols for Nec-1s [ 33]. Seven animal
,41,42]. Oxa12 and Nec-1s
9
,
per group were used. After 30 days, mice were sacrificed in a CO2 chamber followed by
transcardiac perfusion with ice-cold PBS. Brains were then excised, and one hemisphere
was used to isolate the midbrain region, containing the SN, and the striatum, as previously
described [38
,
41], which was rapidly frozen in liquid nitrogen and stored at
−
80 ◦C until
processing for protein extraction. The other hemisphere was fixed in 4% paraformaldehyde
for 48 h and then stored in 20% sucrose/PBS and 0.025% sodium azide, at 4 C, for further
◦
immunohistochemistry analyses.
4.8. Immunohistochemistry
Hemispheres previously fixed in paraformaldehyde were cryoprotected in 20% su-
crose/PBS and embedded in gelatin. Then, sequential coronal brain sections (8
µm thick)
near the midstriatum (Bregma 1.00) and SN (Bregma 3.20) were obtained by cryostat
−
sectioning and mounted on SuperFrost-Plus glas◦s slides (Thermo Fisher Scientific). After-
ward, sections were incubated in warm PBS at 37 C for 15 min, followed by two washes in
PBS, to remove gelatin. Then, sections were blocked in Tris-buffered saline (TBS) contain-
ing 10% (v/v) normal donkey serum (Jackson ImmunoResearch Laboratories Inc., West
Grove, PA, USA) and 0.1% (v/v) Triton X-100 (Sigma-Aldrich) for 1 h. Subsequentially, to
stain dopaminergic neurons, sections were incubated with primary rabbit polyclonal anti-
tyrosine hydroxylase (TH) antibody (#ab112; Abcam, Cambridge, UK, 1:700), overnight
◦
at 4 C. After several washes with PBS, anti-TH primary antibody was detected with
diluted (1:200) Alexa Fluor 488 (anti-rabbit) conjugated secondary antibody (Invitrogen—
Thermo Fisher Scientific) for 2 h at room temperature. After extensive rinsing, sections
were counterstained with Hoechst 33258 (Sigma-Aldrich) and mounted on Mowiol 4-88
(Sigma-Aldrich).
4.9. Image Analysis
Images were obtained by an Axioskop fluorescence microscope (Carl Zeiss GmbH,
Hamburg, Germany). Images were captured from six region-matched sections for nigral
and striatal regions for each animal and converted into a gray scale with an 8-bit format
using the ImageJ software version 1.48 (National Institute of Health, Bethesda, MD, USA).
A threshold optical density was determined for each staining. Areas occupied by positive
staining were quantified in thresholded images, normalized to the total area of interest
region and calculated as percentage of total area.
4.10. Protein Isolation
For total protein isolation, tissues obtained from dissected midbrains and striata were
homogenized in radio-immunoprecipitation assay (RIPA) buffer (50 nM Tris/HCl, pH 8;
150 nM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS) and 1x Halt Protease

Int. J. Mol. Sci. 2021, 22, 5289
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and Phosphatase Inhibitor Cocktail (Pierce, Thermo Fisher Scientific) with a moto-driven
Bio-vortexer (No 1083; Biospec Products, Bartlesfield, UK). Lysates were maintained on ice
for 30 min and were then sonicated and centrifuged at 10,000
were collected and used as total protein extracts. Protein concentrations were determined
×
g for 10 min. Supernatants
by using the Bio-Rad protein assay kit, according to the manufacturer’s instructions.
4.11. Western Blot
Equal amounts of total protein extracts were electrophoretically resolved on 8% SDS-
PAGE. Resolved proteins were then transferred onto nitrocellulose membranes and blocked
with a 5% milk solution in Tris-buffered saline (TBS). Then, membranes were incubated
◦
with primary antibody rabbit polyclonal TH (#ab112, Abcam), overnight at 4 C. After
washing with TBS/0.2% Tween 20 (TBS-T), membranes were incubated with secondary goat
anti-rabbit IgG antibody conjugated with horseradish peroxidase (Bio-Rad Laboratories)
for 2 h at room temperature. Membranes were processed for protein detection using
ImmobilonTM Western (Millipore).
β-actin (AC-15) (#A5441, Sigma-Aldrich) was used
as loading control. Densitometric analysis was performed using the Image Lab Software
version 5.1 Beta (Bio-Rad).
4.12. Statistical Analysis
All data are presented as mean
±
standard error of the mean (SEM). Data analysis were
performed with a one-way analysis of variance (ANOVA) followed by a post hoc Bonferroni
test. Analysis and graphical presentation were conducted with the GraphPad Prim Software
version 8 (GraphPad Software Inc., San Diego, CA, USA). Statistical significance was
achieved when p < 0.05.
Author Contributions: Conceptualization, P.A.D., J.D.A., C.A.M.A. and C.M.P.R.; methodology,
S.R.O., P.A.D., M.M.G., M.B.T.F., C.A.B.R., R.G.P., M.S.E. and M.J.P.; investigation, S.R.O., P.A.D.,
M.M.G., M.B.T.F., C.A.B.R., R.G.P., M.S.E. and M.J.P.; data curation, S.R.O., P.A.D., J.D.A., R.M. and
C.A.M.A.; writing—original draft preparation, S.R.O.; writing—review and editing, S.R.O., P.A.D.,
M.M.G., M.B.T.F., C.A.B.R., R.G.P., M.S.E., M.J.P., R.M., C.A.M.A., J.D.A. and C.M.P.R.; visualization,
S.R.O., J.D.A. and C.A.M.A.; supervision, J.D.A., C.A.M.A. and C.M.P.R.; project administration,
J.D.A. and C.M.P.R.; funding acquisition, C.M.P.R. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received funding from European Structural & Investment Funds through the
COMPETE Program—Programa Operacional Regional de Lisboa—Program Grant LISBOA-01-0145-
FEDER-016405, and from National Funds through the FCT—Fundação para a Ciência e a Tecnologia—
Program Grant SAICTPAC/0019/2015. S.R.O. received a PhD fellowship (PD/BD/128332//2017)
from the FCT.
Institutional Review Board Statement: The study was conducted according to the guidelines of
the Declaration of Helsinki and approved by the institutional Review Board and by the national
authority Direção-Geral de Alimentação e Veterinária (DGAV), initiated on 1 August 2018 (Necrop-
tosis in dopaminergic neurodegeneration in Parkinson’s disease), in accordance with the EU Di-
rective (2010/63/UE), Portuguese laws (DL 113/2013, 2880/2015, 260/2016 and 1/2019) and all
relevant legislation.
Informed Consent Statement: Not applicable.
Acknowledgments: We thank all members of the laboratory for advice and technical assistance.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.

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