α-Linolenic Acid-Valproic Acid Conjugates: Toward Single-Molecule Polypharmacology for Multiple Sclerosis

Article abstract of DOI:10.1021/acsmedchemlett.0c00375

Multiple sclerosis (MS) is a complex inflammatory, degenerative, and demyelinating disease of the central nervous system. Although treatments exist, MS cannot be cured by available drugs, which primarily target neuroinflammation. Thus, it is feasible that a well concerted polypharmacological approach able to act at multiple points within the intricate network of inflammation, neurodegeneration, and demyelination/remyelination pathways would succeed where other drugs have failed. Starting from reported beneficial effects of α-linolenic acid (ALA) and valproic acid (VPA) in MS, and by applying a rational strategy, we developed a small set of codrugs obtained by conjugating VPA and ALA through proper linkers. A cellular profiling identified 1 as a polypharmacological tool able not only to modulate microglia polarization, but also to counteract neurodegeneration and demyelination and induce oligodendrocyte precursor cell differentiation, by acting on multiple biochemical and epigenetic pathways.

Full text of DOI:10.1021/acsmedchemlett.0c00375

pubs.acs.org/acsmedchemlett
Letter
α‑Linolenic Acid−Valproic Acid Conjugates: Toward Single-Molecule
Polypharmacology for Multiple Sclerosis
Michele Rossi,§ Sabrina Petralla,§ Michele Protti, Monica Baiula, Tereza Kobrlova, Ondrej Soukup,
Santi Mario Spampinato, Laura Mercolini, Barbara Monti, and Maria Laura Bolognesi*
Cite This: https://dx.doi.org/10.1021/acsmedchemlett.0c00375
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ABSTRACT: Multiple sclerosis (MS) is a complex inflammatory,
degenerative, and demyelinating disease of the central nervous
system. Although treatments exist, MS cannot be cured by
available drugs, which primarily target neuroinflammation. Thus, it
is feasible that a well concerted polypharmacological approach able
to act at multiple points within the intricate network of
inflammation, neurodegeneration, and demyelination/remyelina-
tion pathways would succeed where other drugs have failed.
Starting from reported beneficial effects of α-linolenic acid (ALA)
and valproic acid (VPA) in MS, and by applying a rational strategy,
we developed a small set of codrugs obtained by conjugating VPA
and ALA through proper linkers. A cellular profiling identified 1 as
a polypharmacological tool able not only to modulate microglia polarization, but also to counteract neurodegeneration and
demyelination and induce oligodendrocyte precursor cell differentiation, by acting on multiple biochemical and epigenetic pathways.
KEYWORDS: Polypharmacology, codrugs, α-linolenic acid, valproic acid
We have contributed to the implementation of polypharma-
cology in the field of neurodegenerative drug discovery.5,6 In
this project, we aimed to harness this knowledge to develop
new polypharmacological tools that deliberately hit multiple
biological targets (i.e., inflammation, neurodegeneration,
demyelination/remyelination) in different brain cell types.
To note, although combination therapy has been advocated
and tested in the clinics for MS,4 to the best of our knowledge,
no polypharmacological approach based on a single
pharmaceutical ingredient, has been reported so far.
To do so, we turned our attention to the development of
versatile polypharmacological tools, i.e., codrugs.7 “Codrugs”
or “mutual prodrugs” are single molecules obtained by the
conjugation of two therapeutic compounds with synergistic
activity, via a cleavable linker.8,9 A marketed example is the
antibacterial sultamicillin, an ampicillin/sulbactam codrug
(Figure S1). The fact that the two starting compounds,
following metabolic transformation, have the potential to be
released in the same target cells and at the same time is a
peculiar feature of codrugs with respect to combinations (two
ultiple sclerosis (MS) is a chronic complex malady of
Mthe central nervous system (CNS) characterized by
inflammation, demyelination, and neurodegeneration, resulting
in a progressively increasing disability.1 With the only
exceptions of ocrelizumab and siponimod, which have a
moderate effect on disease progression, none of the 17
currently available drugs are able to halt or at least slow the
relentless neuronal disability.2 Indeed, current therapies,
focused primarily on pathological immune responses (i.e.,
immunomodulation and immunosuppression), are generally
less effective in the progressive forms of the disease than in the
relapsing remitting ones.2 Therefore, curing demyelination and
the concomitant neurodegeneration, in addition to immuno-
modulation, needs to be addressed for a truly curative effect in
all patients.3
Developing such treatment is not an easy task, as it involves
targeting complex pathological cascades in different brain cell
types. Therefore, while monotherapies may be inappropriate,
multipronged approaches will likely be needed to support the
wide range of CNS functions and minimize injury and toxicity
involving neurons, oligodendrocytes, and microglia.4 On this
basis, we were motivated to develop a polypharmacological
treatment able not only to modulate immunomodulatory/
inflammatory aspects of the disease, but also to counteract
neurodegeneration and demyelination and to sustain remyeli-
nation.
Received: July 7, 2020
Accepted: October 23, 2020
https://dx.doi.org/10.1021/acsmedchemlett.0c00375
© XXXX American Chemical Society
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single compounds, each one with an individual pharmacoki-
netic profile).9,10 This potentially concomitant delivery appears
particularly advantageous from a polypharmacology perspec-
tive.9,10 On the other hand, it should be mentioned that the
codrug approach is applicable only to starting compounds
carrying functional groups suitable for conjugation.8
We focused on the development of codrugs between
valproic acid (VPA) and α-linolenic acid (ALA), both
amenable to linking/conjugation (Figure 1). We reasoned
Figure 2. Design of ALA-VPA conjugates 1−4.
On the basis of previous studies,9,17 we expected 1−4 to be
stable in the circulation at different extents, depending on the
nature of the formed bonds (amide vs ester). Then, hydrolysis
should occur in the brain tissues where specific enzymes able
to hydrolyze endogenous fatty acid conjugates are present. As
reported for edasalonexent9 and a fatty acid cysteamine
conjugate,17 the metabolism of 1−4 might be mediated by
endocannabinoid metabolic enzymes, including fatty acid
amide hydrolase (FAAH), monoacylglycerol lipase, and N-
acylethanolamine acid amidase. Then, each released starting
framework (VPA or ALA) should maintain the ability to
recognize its specific targets and, collectively, to produce
multiple, synergistic pharmacological effects. In principle, the
same effects could be also reached by a combination treatment
of VPA and ALA. However, according to the previous
reports,9,17 the herein proposed single-molecule conjugates
might have advantages with respect to a classical drug
combination: VPA and omega-3 FAs have the potential to
be delivered in the target cells at the same time and in
equimolar concentrations. Since multiple biological pathways
in multiple brain cell types might be simultaneously hit, it is
feasible to hypothesize that the resulting polypharmacology of
1−4 could be peculiar and not be replicated by administering
VPA and ALA in combination, where each one has an
individual ADME (absorption, distribution, metabolism and
excretion) profile.
In the following, the synthesis, preliminary pharmacokinetic
evaluation, and biological characterization of 1−4 at a cellular
level are reported. We believe that, at this initial stage, cell-
based systems maintain a reasonable screening efficiency while
preserving network interactions, critical in a polypharmacology
context.
Chemistry. For the synthesis of conjugates 1−4, a linear
synthetic strategy based on 1-ethyl-3-(3-(dimethylamino)-
propyl)carbodiimide (EDC)/4-dimethylaminopyridine
(DMAP) coupling and protection/deprotection steps was
followed (Scheme 1). First, the three bifunctional linkers, i.e.,
ethylene glycol, ethanolamine, and ethylenediamine, were
monoprotected with the suitable protecting group to allow
selective reaction on the proper function.
Figure 1. Chemical structures of α-linolenic acid, docosahexaenoic
acid, edasalonexent, valproic acid, and salicylic acid.
that starting from a marketed drug (VPA) and an omega-3
polyunsaturated fatty acid food supplement (ALA) could
mitigate the risk of toxicity for use in humans, thereby
potentially expediting the clinical translation of eventual
candidates.
Our strategy was founded upon the reported beneficial
effects of ALA and VPA in MS. Several studies have
demonstrated that ALA supplementation delays onset and
reduces demyelination and cognitive dysfunction in the
experimental autoimmune encephalomyelitis (EAE) model.
These positive effects are accompanied by a shift in microglial
polarization toward the beneficial M2 phenotype.11 In
addition, higher levels of ALA were associated with lower
disease activity.12
Similarly, VPA has been shown to be effective in animal
models of the disease.13 VPA, used for the treatment of
neurological disorders for more than 40 years, exerts its
therapeutic effects through multiple mechanisms. Among
them, which include GABA, glutamate receptor and sodium
and calcium voltage-gated channel modulation, its activity as a
histone deacetylase (HDAC) inhibitor has recently come to
light.14 As an HDAC inhibitor, VPA is reported to be
neuroprotective and neuroregenerative in various neurological
diseases,14 including MS (although some controversy does
exist).15
Thus, we embarked on the development of ALA-VPA
codrugs for MS. We deemed that such a concerted,
simultaneous modulation of multiple critical pathways by
VPA and ALA can result in a truly immunomodulatory,
neuroprotective, and neurorestorative effect.
Standard reactions with di-tert-butyl decarbonate (Boc2O)
or tert-butyldimethylsilyl chloride (TBSCl) provided the N-
Boc- or the O-TBS-protected linkers 5−8 in very good yields
(87−98%). Monoprotected 5−8 were then coupled with VPA
after activation with EDC/DMAP to provide ester or amide
intermediates 9−12, in moderate to good yields (42−68%).
Treatment with trifluoracetic acid (TFA) or tetrabutylammo-
nium fluoride (TBAF) removed the Boc- or TBS-protecting
groups, respectively, providing intermediates 13−16 (89−
98%). Both deprotection reactions were conducted under
anhydrous conditions and at 0 °C, in order to avoid
Design. The codrug strategy was based on a set of linkers
(ethylene glycol, ethanolamine, ethylenediamine) that allowed
VPA and ALA to be covalently joined via their carboxylic acid
functions. This linker strategy has been recently validated by
the development of edasalonexent,9 a conjugate of salicylic acid
and docosahexaenoic acid (DHA, Figure 1), currently clinically
evaluated to treat Duchenne muscular dystrophy.16 The linking
strategy allowed the formation of ester or amide bonds
between the acid functions of VPA and ALA, obtaining
conjugates 1−4 (Figure 2).
B
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a
(viability > 80% at 25 μM; Table S1). Hence, we selected 1
and 2 for further studies, because they are representative of
two different linker types and are the less toxic ones among the
synthesized set, especially at the more therapeutically relevant
concentrations (5 and 10 μM).
Scheme 1. Synthesis of PUFA−VPA Conjugates 1−4
Plasma Stability Assay. Our aim was to develop plasma-
stable conjugates that would be hydrolyzed in the brain.
Selective localized hydrolysis of conjugates 1 and 2 would
permit a CNS-targeted profile and less side effects. Thus,
plasma stability is an important pharmacokinetic prerequisite
for 1 and 2. As reported by stability assays performed by LC-
DAD-MS/MS analysis (Figure 3), conjugates 1 and 2 are
a
Reagents and conditions: (i) TBSCl, imidazole, DCM, r.t., 24 h; (ii)
Boc2O, DCM, r.t., 24 h. (iii) EDC, DMAP, DCM, r.t., 8 h, N2; (iv)
TFA, DCM, r.t., 2 h; (v) TBAF, r.t., 24 h; (vi) ALA, EDC, DMAP,
DCM, r.t., 8 h, N2.
concomitant VPA hydrolysis. Finally, targets 1−4 were
synthesized from ALA and the respective VPA-functionalized
linker (13−16), using the previous EDC/DMAP protocol.
However, in this case, the reaction was carried out under
nitrogen atmosphere and in the dark, in order to minimize
oxidation side-reactions.
Neuro- and Hepatotoxicity Assays. A preliminary
cytotoxicity screening was performed on conjugates 1−4
using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide) assay (Tables 1 and S1). Increasing
Table 1. Effect of 1−4 and Parent Compounds VPA and
ALA on Cell Survival/Death in CGNs, As Determined by
MTT Assay after 24 h Treatment
Figure 3. Rat plasma stability of conjugates 1 and 2 at 37 °C:
percentage remaining upon incubation, assessed by means of LC-
DAD-MS/MS. Analyses were performed in triplicate.
a
% mean SE
compd
0 [μM]
5 [μM]
10 [μM]
25 [μM]
50 [μM]
VPA
ALA
1
2
3
4
100
100
100
100
100
100
6
100 15
93 13
98 18
97 16
119 17
118 16
106
97
107
110
116
114
3
3
3
3
3
3
85 14
79 12
89 14
96 15
99 17
97 17
45 5***
99 15
85 14
41 6***
45 8***
b
stable for more than 85% after 1 h of incubation at 37 °C in rat
plasma. Specifically, the percentage of diamide 1 remaining
upon incubation is higher than that of ester/amide 2 (89% for
1 and 85% for 2). The mean plasma remaining fraction of the
two conjugates after 1 h of incubation was compared by means
of t test and a statistically relevant difference (p < 0.01) was
observed.
In Vitro BBB Permeation Assay. In parallel, we tested 1
and 2 in the blood-brain barrier (BBB) specific parallel artificial
membrane permeability assay (PAMPA-BBB) (Table 2). Both
compounds were predicted to be BBB-permeable. Conjugate 1
had an effective permeability (Pe) of 27.98, which placed it in
6
6
6
6
6
b
b
71
8
a
Results are expressed as percentages of controls and are the mean
SE of at least 3 independent experiments, each run in triplicate. ***p
< 0.001 compared to control conditions (0 μM), Student’s t test.
b
concentrations (5, 10, 25, 50 μM) of each conjugate and
parent compound (VPA and ALA) were tested for 24 h in
primary rat cerebellar granule neurons (CGNs) and the human
liver carcinoma immortalized cell line (HepG2). Drug toxicity
is one of the significant shortcomings in CNS drug discovery.18
Furthermore, drug-induced liver injury has been associated
with a number of MS drugs, including disease-modifying and
symptomatic therapies.19
From Table 1, it is evident that diamide 1 and ethanolamide
2 display a low neurotoxicity profile, and also at the higher
tested concentrations (viability >85% at 50 μM). They are less
toxic compared to the inverse ethanolamide (3, 41% viability)
and diester (4, 45%) analogues and also to reference
compounds. Notably, while the CNS drug VPA shows
negligible neurotoxicity, ALA is neurotoxic at 50 μM (45%).
In addition, both diamide 1 and ethanolamide 2 showed a low
hepatotoxicity, even at the higher tested concentrations
Table 2. PAMPA Effective Permeability (Pe) Values with
Related Predictive BBB Penetrations of Commercial Drugs,
1 and 2
BBB penetration estimation
a
compd
Pe SEM (× 10−6cm s−1
)
CNS (+/−)
1
2
27.98 5.30
14.59 1.61
0.19 0.07
0.35 0.31
21.93 2.06
5.96 0.59
CNS +
CNS +
CNS −
CNS −
CNS +
CNS +
furosemide
ranitidine
donepezil
tacrine
a
Pe SEM (n = 3). Each compound was assessed in quadruplicate.
C
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the high BBB permeability category (Table 2), higher than that
of two CNS drugs (donepezil and tacrine).
Brain Stability Assay. We anticipated that, once they
entered into the brain, conjugates would be hydrolyzed to the
individual components by intracellular metabolic enzymes
processing fatty acid derivatives (e.g., FAAH).9,17 Thus, the
stability of 1 and 2 was evaluated by LC-DAD-MS/MS analysis
of rat brain homogenate incubated with the conjugates, which
was sampled at regular intervals over 1 h. To obtain initial
clues on the implication of FAAH enzymes in hydrolysis, the
assays were performed in the absence and presence of 5 μM
FAAH inhibitor PF-3845.9
As shown in Figure 4, both conjugates undergo metabolism
(i.e., total disappearance of starting compound) within 1 h of
Figure 5. Effect of 1, 2, and VPA on HDAC inhibition, evaluated
through analysis of acetylated histone levels in medulloblastoma
(DAOY) cells. Densitometric analysis of the bands is shown (mean
SD of three independent experiments); the amount of AcH3 is
normalized to that of totH3. *p < 0.05; **p < 0.01; ***p < 0.001 vs
vehicle (Newman−Keuls test after ANOVA).
panel). Remarkably, a similar increase of H3 acetylation was
detected in neuronal cells exposed to conjugates 1 and 2 at 0.5
mM (Figure 5, right panel).
Immunomodulation Assays. Microglia, essential regu-
lators of neuroinflammatory processes, can be polarized to two
distinct activation states: M1 and M2. M1 microglia have
proinflammatory, neurotoxic properties and are modeled in
vitro through lipopolysaccharides (LPS) activation. Alterna-
tively, M2 microglia are involved in anti-inflammatory
processes, contributing to trophic support of neurons and
neuroprotective functions. A marked microglial activation with
high expression levels of proinflammatory genes is found in MS
lesions.23 Conversely, the neuroprotective effects of the MS
drug glatiramer are mediated by activated M2 microglia.24 To
this end, we evaluated whether 1 and 2 modulate microglial
shift from an M1 neurotoxic phenotype to an M2 neuro-
protective one. Therefore, N9 microglial cells were treated
with 100 ng/mL LPS in the presence or absence of increasing
concentrations (0.5, 1.5, 10 μM) of 1, 2, parent compounds
VPA and ALA, as well as and their 1:1 combination (ALA
+VPA, Figure 6). After pretreatment for 6 h and administration
for a further 24 h, the microglial phenotype was evaluated
through Western blot analysis of the M1-iNOS (inducible
Nitric Oxide Synthase) and M2-TREM2 (Triggering Receptor
Expressed on Myeloid cells 2) markers (Figure 6). We also
assessed nitrite production, due to iNOS induction, in the
incubation media.
As reported in Figure 6, compounds 1 and 2, even at low
concentration (0.5 μM), significantly decrease microglia
production of the M1 proinflammatory marker iNOS, at an
extent similar to that of parent compounds. Remarkably, they
were more effective than the ALA+VPA combination.
Interestingly, 1 and 2 display immunomodulatory properties,
as revealed by the unchanged expression of TREM2 (M2
marker). This is important, as TREM2 is reported to invoke
microglia to phagocytose myelin debris in MS lesions25 and its
blockage results in an amplified demyelination in EAE.26
Regarding the levels of nitrites, 1 and 2 reduced their
concentration more effectively than VPA and similarly to the
ALA+VPA combination.
Figure 4. Hydrolysis kinetics in rat brain lysate (37 °C). Percentages
of 1 or 2 remaining over time upon incubation in the absence and
presence of FAAH inhibitor PF-3845, assessed by means of LC-DAD-
MS/MS. Analyses were performed in triplicate.
incubation, with a more marked decrease rate with regard to
ethanolamide 2 (half-life of about 16 min) compared to
diamide 1 (half-life of about 23 min). Moreover, we can infer
that the observed hydrolysis is FAAH-dependent, as the
addition of PF-3845 blocks the hydrolysis of both 1 and 2 to a
large extent. To note, although it appears that the plasma
stability of these codrugs is higher than that in rat brain
homogenates, we cannot exclude that hydrolytic reactions
might occur in tissues like the liver.
After getting a preliminary indication that conjugates 1 and
2 would be metabolized to afford the parent compounds
selectively in brain target cells, we investigated the biological
effects that they could induce.
HDAC Assays. Several studies have shown that long-term
administration of VPA results in regulation of inflammatory
processes, neuroprotection, and cell differentiation through
HDAC inhibition.20,21 Thus, we preliminarily verified whether
1 and 2 would retain the HDAC inhibitory capacity of the
parent VPA in human medulloblastoma cell line DAOY
(Figure 5). VPA is a relatively weak HDAC inhibitor, with
millimolar activity (a clinically achievable concentration).22
Thus, we tested it in the 0.1−1.5 mM range.
Oli-Neu Cell Cytotoxicity Assays. Oligodendrocytes are
the myelin-forming cells. They are the end product of a cell
lineage which, following finely tuned cycles of proliferation,
migration, and differentiation, finally produces myelin.27 The
As expected, VPA treatment induced a strong inhibition of
HDAC, shown by a significant, concentration-dependent
increment in acetylation levels of histone H3 (Figure 5, left
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decrease cell viability already at 1 μM, conjugates 1 and 2 show
no toxicity even at the higher concentrations (10 μM).
Intriguingly, cells treated with ethanolamide 2 revealed a
significantly higher metabolic activity (MTT readout)
compared with control cells (Table 3).
Oli-Neu Cell Proliferation and Differentiation Assay.
OPC proliferation and migration to the CNS injury site as well
as their differentiation are critical steps toward remyelination in
MS. HDAC activity plays a crucial role in OPC proliferation/
differentiation; therefore, HDAC modulation is widely
considered a promising therapeutic target for MS.30 Accord-
ingly, VPA has been shown to increase myelin repair in the
EAE model, by acting on OPC recruitment.31 Therefore, we
tested the effect of our compounds on OPC proliferation and
differentiation, through cell counting, measure of filaments,
and analysis of proliferation/differentiation markers (Figure 7).
Figure 6. Immunomodulatory effects of conjugates 1 and 2, ALA,
VPA, and their 1:1 equimolar combination (ALA+VPA) in microglial
cells evaluated through Western blot analysis of iNOS and TREM2
expression, as well as the indirect extent of released NO trough nitrite
measurement in the medium. GAPDH was used as loading control.
Results are expressed as percentage of controls and are the mean SE
of at least two independent experiments. **p < 0.01; *p < 0.05
compared to control conditions (0 μM), one way-ANOVA followed
by Bonferroni’s posthoc test.
inhibitory microenvironment in MS lesions abolishes the
expansion and differentiation of resident oligodendrocyte
precursor cells (OPCs) into mature myelin-forming oligoden-
drocytes. Loss of myelin, in turn, accounts for motor and
cognitive deficits in MS patients. Thus, chemical manipulation
of OPC fate may open new avenues for regenerative therapies
in MS.28
Here, we used Oli-neu cells, an oligodendroglial precursor
cell line, as an accepted in vitro model to screen the
remyelination potential of our molecules.29 First, we tested
the toxicity of our conjugates and parent compounds against
Oli-Neu. After treatment with increasing concentrations (1−10
μM) for 24 h, the MTT assay shows that, while VPA and ALA
Figure 7. Effect of conjugates 1 and 2, ALA, VPA, and their 1:1
equimolar combination (ALA+VPA) on proliferation and differ-
entiation of Oli-Neu cells at 5 μM through cell counting (A, B)
measure of filaments length (A, C) at 48 h, as well as expression of
Olig2 at 48h (D, E) and CNPase (D, F) at 72 h. Results are expressed
as percentage of controls and are the mean
SE of at least three
different experiments. **p < 0.01; *p < 0.05 compared to control
conditions (0 μM), one way-ANOVA followed by Bonferroni’s
posthoc test.
Table 3. Effect of 1 and 2 on Cell Survival/Death through MTT Assay in Oli-Neu Cells (Immortalized Mouse Oligodendrocyte
Precursor Cells), in Comparison with Parent Compounds ALA and VPA
a
% mean SE
compd
0 [μM]
0.5 [μM]
1 [μM]
5 [μM]
10 [μM]
b
b
b
b
b
b
b
b
VPA
ALA
1
100
100
100
100
6
75 7**
78 5**
72 6**
74 9**
71 8**
88 5*
76 7**
68 6**
93 6**
6
6
6
75
95
9
8
c
89
9
4
b
2
110
115 7**
105
8
101
8
a
b
Results are expressed as percentages of controls and are the mean SE of at least 2 independent experiments, each run in triplicate. **p < 0.01.
c
*p < 0.05 compared to control conditions (0 μM), one way-ANOVA followed by Bonferroni’s posthoc test.
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While Oli-Neu cells treated with VPA+ALA show a significant
increase in the cell number, with a parallel decrease in the
filament length, compound 1 and 2, similarly to VPA, decrease
cell number with a parallel increase in filament length. This
suggests that 1 and 2 might induce OPCs differentiation to
oligodendrocytes. To further support this hypothesis, Western
blot analysis showed a marked decrease in the expression of
Olig2, a marker of OPCs proliferation, for 1, 2 and VPA, but
not for ALA and ALA+VPA. This profile was paralleled by a
significant increase in the expression of CNPase, a marker of
differentiation, for both 1 and VPA. Collectively, these data
point to an induction of OPC differentiation toward
oligodendrocytes by 1 and 2, which, similarly to VPA, can
perform myelination/remyelination. Importantly, this behavior
is not shown by ALA and neither by ALA+VPA combination
but is peculiar to conjugate 1.
Neuroprotection Assay. Several lines of evidence point to
MS as not only an inflammatory but also a neurodegenerative
disease. Thus, neuroprotective agents may hold promise for
MS therapy.32 VPA has been reported to exert neuroprotective
effects and to reduce glutamatergic excitatory neurotransmis-
sion.33 Thus, to test the neuroprotective capability of 1 and 2,
we used a glutamate excitotoxicity model of primary cultural
neurons. At day 8 in vitro (8 DIV), CGNs were pretreated
with the compounds for 6 h, before adding 100 μM glutamate/
10 μM glycine insult for further 24 h. As illustrated in Figure 8,
that of the parent compounds, as well as to their equimolar
combination. The collected results, the experience gained from
the use of VPA in humans for over 40 years, and the fact that
ALA is a natural product present in many foods might warrant
further investigation into this series. In parallel, the fact that a
recent study has highlighted a dampened remyelination and a
decreased oligodendrocyte cell count for VPA34 and its use in
MS therapy is still controversial15 suggests that other
conjugations of promising compounds should be explored.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.0c00375.
Experimental details for chemistry and biological assays,
cell toxicity plots, NMR spectra, LC-MS/MS chromato-
grams and MS spectra for compounds 1 and 2 (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Maria Laura Bolognesi − Department of Pharmacy and
Biotechnology, Alma Mater Studiorum - University of Bologna,
I-40126 Bologna, Italy; orcid.org/0000-0002-1289-5361;
Email: marialaura.bolognesi@unibo.it
Authors
Michele Rossi − Department of Pharmacy and Biotechnology,
Alma Mater Studiorum - University of Bologna, I-40126
Bologna, Italy
Sabrina Petralla − Department of Pharmacy and Biotechnology,
Alma Mater Studiorum - University of Bologna, I-40126
Bologna, Italy
Michele Protti − Department of Pharmacy and Biotechnology,
Alma Mater Studiorum - University of Bologna, I-40126
Bologna, Italy; orcid.org/0000-0001-9310-4957
Monica Baiula − Department of Pharmacy and Biotechnology,
Alma Mater Studiorum - University of Bologna, I-40126
Bologna, Italy; orcid.org/0000-0003-0363-0633
Tereza Kobrlova − Biomedical Research Center, University
Hospital, CZ-500 05 Hradec Kralove, Czech Republic
Ondrej Soukup − Biomedical Research Center, University
Hospital, CZ-500 05 Hradec Kralove, Czech Republic
Santi Mario Spampinato − Department of Pharmacy and
Biotechnology, Alma Mater Studiorum - University of Bologna,
I-40126 Bologna, Italy
Laura Mercolini − Department of Pharmacy and Biotechnology,
Alma Mater Studiorum - University of Bologna, I-40126
Bologna, Italy; orcid.org/0000-0002-0644-9461
Barbara Monti − Department of Pharmacy and Biotechnology,
Alma Mater Studiorum - University of Bologna, I-40126
Bologna, Italy; orcid.org/0000-0003-0330-482X
Figure 8. Neuroprotective effect of 1 and 2, ALA, VPA, and their 1:1
equimolar combination (ALA+VPA) on glutamate/glycine excitotox-
icity through MTT assay in differentiated CGNs (8 DIV) pretreated
for 6 h and cotreated for a further 24 h at 5, 10, and 25 μM. Results
are expressed as percentage of controls and are the mean SE of at
least four different experiments, each run in triplicate. *p < 0.05, **p
< 0.01, ***p < 0.001 vs control, #p < 0.05, ##p < 0.01, ###p < 0.001 vs
Glu/Gly, green asterisk **p < 0.01, green asterisk ***p < 0.001 vs
ALA+VPA 1:1, blue asterisk **p < 0.01 vs ALA, one way-ANOVA
followed by Bonferroni’s posthoc test..
pretreatment with 1 resulted a more neuroprotective against
glutamatergic excitotoxicity when compared with VPA, ALA
VPA+ALA 1:1, and 2, especially at lower concentrations (5
and 10 μM). Furthermore, while reference compounds (alone
and in combination) increase glutamate/glycine excitotoxicity
at higher doses, 1 was not toxic even at the highest
concentration tested (25 μM).
Complete contact information is available at:
https://pubs.acs.org/10.1021/acsmedchemlett.0c00375
CONCLUSIONS
■
Although significant progress has been made in developing
immunomodulatory treatments, there is a lack of therapeutic
options that address the multiple, concomitant pathophysio-
logical aspects of MS. This study, albeit very preliminary,
demonstrates for the first time the potential of VPA/ALA
codrugs to achieve polypharmacology in MS. Particularly,
diamide conjugate 1 displays an overall immunomodulatory,
remyelinating, and neuroprotective profile that is superior to
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Author Contributions
§M.R. and S.P. equally contributed to this work.
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Funding
This work was supported by the University of Bologna (Grant
RFO 2016). M.R. acknowledges the Emilia Romagna regional
Ph.D. program “Piano triennale Alte competenze per la ricerca,
̀
il trasferimento tecnologico e l’imprenditorialita” (Ph.D. fellow
2016−2019). O.S and T.K. acknowledge the University
H o s p i t a l H r a d e c K r a l o v e ( M H C Z - D R O )
(UHHK,00179906) and (CZ.02.1.01/0.0/0.0/18_069/
0010054) ERDF project.
Notes
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antiepileptic drug valproic acid restores T cell homeostasis and
ameliorates pathogenesis of experimental autoimmune encephalo-
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(14) Monti, B.; Polazzi, E.; Contestabile, A. Biochemical, molecular
and epigenetic mechanisms of valproic acid neuroprotection. Curr.
Mol. Pharmacol 2009, 2 (1), 95−109.
The authors acknowledge “Multi-target paradigm for innova-
tive ligand identification in the drug discovery process
(MuTaLig)” COST Action CM15135.
̈
(15) Nielsen, N. M.; Svanstrom, H.; Stenager, E.; Magyari, M.;
Koch-Henriksen, N.; Pasternak, B.; Hviid, A. The use of valproic acid
and multiple sclerosis. Pharmacoepidemiol. Drug Saf. 2015, 24 (3),
262−8.
ABBREVIATIONS
■
ALA, α-linolenic acid; BBB, blood-brain barrier; CGNs,
cerebellar granule neurons; CNS, central nervous system;
DHA, acid docosahexaenoic acid; DMAP, 4-dimethylamino-
pyridine; Eight DIV, day 8 in vitro; EAE, experimental
autoimmune encephalomyelitis; EDC, 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide; FAAH, fatty acid
amide hydrolase; HDAC, histone deacetylase; iNOS, inducible
nitric oxide synthase; MS, multiple sclerosis; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OPCs,
oligodendrocyte precursor cells; PAMPA, parallel artificial
membrane permeability assay; TBAF, tetrabutylammonium
fluoride; TFA, trifluoracetic acid; TREM2, triggering receptor
expressed on myeloid cells 2; VPA, valproic acid.
(16) Finanger, E.; Vandenborne, K.; Finkel, R. S.; Lee Sweeney, H.;
Tennekoon, G.; Yum, S.; Mancini, M.; Bista, P.; Nichols, A.; Liu, H.;
Fretzen, A.; Donovan, J. M. Phase 1 Study of Edasalonexent (CAT-
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Muscular Dystrophy. J. Neuromuscul Dis 2019, 6 (1), 43−54.
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Bordwell, C.; Sewell, A.; Nichols, A. J.; Chandran, S.; Lonkar, P.;
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Cysteamine Conjugates as Novel and Potent Autophagy Activators
That Enhance the Correction of Misfolded F508del-Cystic Fibrosis
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