Promising Non-cytotoxic Monosubstituted Chalcones
to Target Monoamine Oxidase-B
A library of monosubstituted chalcones (1–17) bearing electron-donating and electron-withdrawing groups on both aromatic rings were selected. The cell viability on human tumor cell lines was evaluated first. The compounds unable to induce detectable cytotoxicity (1, 13, and 14) were tested using the monoamine oxidase (MAO) activity assay. Interestingly, they inhibit MAO-B, acting as competitive inhibitors, with 13 and 14 showing the best profiles. In particular, 13 exhibited a potency higher than that of safinamide, taken as a reference. Docking studies and crystallographic analysis showed that in human MAO-B 13 binds with the halogen-substituted aromatic ring in the entrance cavity, similar to safinamide, whereas 14 is accommodated in the opposite way. The main conclusion of this cell biology, biochemistry, and structural study is to highlights 13 as a chalcone derivative that is worth consideration for the development of novel MAO-B-selective inhibitors for the treatment of neurodegenerative diseases.
<!>Author Contributions<!>
<p>Chalcones are a key structure
motif within the kingdom Plantae and are widely found in many edible
plants.1 They are open-chain flavonoids
in which a three-carbon α,β-unsaturated carbonyl moiety
joins the two aromatic rings and are considered as open-chain intermediates
in the synthesis of flavones.2 This peculiar
chemical structure endows chalcones with the capability to hit different
targets (enzymes and receptors) and to exert a variety of biological
activities. The chalcone skeleton is therefore widely used in medicinal
chemistry and drug discovery3,4 to obtain derivatives
with anticancer, antioxidant, antiviral, and anti-inflammatory activities
and for the treatment of neurodegenerative disorders.5−9</p><p>Chalcone derivatives exert their anticancer activity through
multiple
mechanisms involving different targets,8,10 and various
targets are also responsible for the effect of chalcone analogues
on neurodegenerative diseases.11−13 In this latter field, monoamine
oxidases (MAOs) play an important role, being mitochondrial and flavin
adenine dinucleotide (FAD) cofactor-dependent enzymes, which catalyze
the oxidative deamination of monoamine neurotransmitters.14 In particular, the MAO-B isoform is mainly investigated
for treatments of neurodegenerative disorders (including Parkinson's
and Alzheimer's diseases), while the MAO-A isoform mostly deals
with neurological and psychiatric disorders.14−16 In this connection,
although a number of MAO-A and MAO-B inhibitors are already available
in clinical practice, the need to reduce their side effects and/or
to improve their selectivity is still definitely of interest and encourages
studies aimed at developing novel types of inhibitors.</p><p>The chalcone
structure has been considered an interesting non-nitrogen-containing
scaffold.17−20 In a recent paper, detailed structure–activity relationships
(SAR) were discussed for the inhibitory activity and selectivity toward
MAOs, with regard to the influence of different substituents on both
aromatic rings of chalcone derivatives and compounds containing the
chalcone motif.17 In particular, the introduction
of heterocycles, the presence of nitro groups or lipophilic electron-donating
groups, and halogen substitution on phenyl ring A and/or B are some
of the modifications that were designed for the chalcone scaffold,
and frequently, more than one modification/substitution was introduced
on the same compound.17 On the basis of
literature data, among the simplest, less substituted chalcone derivatives,
the most active compounds against the MAO-B isoform are characterized
by the presence of hydroxyl and methoxy substituents at ortho and para positions, respectively, of the A aromatic
ring and a chlorine atom at the para position of
the B ring. Nevertheless, a role for each specific substituent did
not emerge.12,13,17,18,21</p><p>In this
frame, we focused our attention on a small library of monosubstituted
chalcone derivatives bearing an electron-donating or electron-withdrawing
substituent in the A or B ring, with the aim to further clarify the
function of the single substituent in the biological properties. Such
derivatives were previously reported and studied as antiproliferative
agents on leukemia cells,22 and for them
few or no data on MAO activity were reported.19,23 All of the compounds were synthesized using a classical Claisen–Schmidt
condensation of the appropriate acetophenone with the corresponding
aldehyde in a methanolic solution of sodium hydroxide. The products
were obtained after being recrystallized twice from ethanol with a
good grade of purity (see the Supporting Information). As the absence of cytotoxicity is a mandatory requirement for
compounds to be used to treat neurodegenerative diseases, the potential
antiproliferative effects of the compounds belonging to the library
were evaluated on a wider number of human tumor cell lines. The chalcone
derivatives that were unable to induce any significant antiproliferative
effect were selected and further studied as potential inhibitors for
human recombinant MAO-A and MAO-B. In detail, we evaluated the inhibition
constant values, the selectivity indices, and the mechanism of inhibition.
The kinetic characterization of the most interesting derivatives, 13 and 14, integrated with docking studies and
crystallographic experiments uncovered their binding mode inside the
MAO-B active site. Overall, our findings expand the knowledge in terms
of possible molecular interactions between the chalcone scaffold and
MAO-B and highlight 13 as a scaffold worth further study
in view of the development of new promising drugs for neurodegenerative
diseases.</p><p>The absence of any cytotoxicity and the enzyme inhibition
mechanism
are fundamental elements in the development of novel MAO-B inhibitors
to target neurodegenerative diseases. With this aim, cell biology,
biochemical, and structural studies were combined to explore the activity
profiles of a small library of monosubstituted chalcones as potential
MAO-B inhibitors.</p><p>Table 1 reports
the chalcone series and the results on antiproliferative activity,
which was evaluated through an in vitro assay performed
on three tumor cell lines, i.e., A2780 (ovarian carcinoma),
HT-29 (colorectal adenocarcinoma), and MSTO-211H (biphasic mesothelioma).
Data are expressed in terms of the GI50 value, i.e., the concentration of compound able to inhibit 50%
cell growth with respect to the control culture. Unsubstituted chalcone
and safinamide were reported as reference.</p><p>Values are reported as mean ±
SD of at least three independent experiments in duplicate.</p><p>Interestingly, these results show
that derivatives 1, 13, and 14 were unable to induce any
cytotoxicity in any of the tested cell lines. Otherwise, the majority
of the chalcones belonging to the selected library, as for the unsubstituted
chalcone scaffold, induced a significant antiproliferative effect,
with GI50 values ranging from 2.8 to 16.7 Î¼M. In
particular, in the series 1–9 bearing
an electron-donating or electron-withdrawing R2 substituent,
derivative 2, characterized by the m-NO2 group, showed the highest cytotoxic effect in all
of the considered cell lines (GI50 values ranging from
2.8 to 5.1 Î¼M), while the o-NO2 analogue 1 was completely ineffective on cells. For all of the other
derivatives (3–9), an intermediate
effect was observed.</p><p>With regard to 10–17, interesting
differences in antiproliferative activity were observed for the derivatives
bearing CF3 as the R1 substituent (12–14). In detail, while the o-CF3-substituted chalcone 12 was able to
induce significant cytotoxicity, the insertion of the same group at
the meta (13) or para (14) position abolished the effect on cells. For 10–11 and 15–17, the position of the NO2 or OCH3 substituent,
respectively, did not significantly affect the cytotoxicity. Finally,
as expected, safinamide was completely ineffective on human cancer
cells.</p><p>On the basis of the antiproliferative activity data (Table 1), the compounds devoid
of detectable
cytotoxic activity (GI50 > 20 Î¼M) on all of the
considered
cell lines, i.e., 1, 13, and 14, were selected and evaluated as potential inhibitors
for human recombinant MAO-A and -B enzymes using kynuramine (kyn)
as the substrate. Moreover, the most cytotoxic derivative 2, unsubstituted chalcone, and compound 17, which had
been previously studied,19,23 were also tested as
reference compounds. Additionally, safinamide24 and isatin25 were included in the study
as reversible standard inhibitors of MAOs.</p><p>All of the tested
chalcones were found to inhibit both MAO-A and
MAO-B. In particular, the Lineweaver–Burk double-reciprocal
plots (1/v0 vs 1/[S]) of the kinetic data
for MAO-B in the presence of various concentrations (5–40 nM)
of 13 (Figure 1A) clearly demonstrate a competitive mode of inhibition, as
only the x-intercept (−1/Km) is affected by the presence of the compound, in contrast
to the y-intercept (1/Vmax), which is not significantly different at all tested concentrations.
The reversibility of the inhibition was evaluated by incubating MAOs
with 13 (at a concentration equal to 4 times the Ki value) for 20 min before starting dialysis
cycles. After dialysis, the MAO activity was fully recovered, supporting
the reversibility of the mode of inhibition of this compound. The
same behavior was found for 1 and 14, as
can be clearly seen in Figure 1B, where results for the lead compound (chalcone) and safinamide
are also shown. This competitive behavior has been reported previously
for other chalcone derivatives.20,23</p><p>Competitive inhibition
of chalcone derivatives on human MAO-B activity.
Lineweaver–Burk double-reciprocal plots of MAO-B initial velocity
vs substrate concentration (1/v0 vs 1/[kynuramine])
in the absence and presence of various concentrations of compounds.
Continuous lines are the results of linear regression analysis of
plotted data (r > 0.98). (a) Reciprocal plots
at
different concentrations of 13 (5–40 nM) showing
that increasing the concentration of the compound increases the effect
on Km (from the x-intercept),
while no effect on Vmax (from the y-intercept) is observed. (b) Examples of reciprocal plots
in the presence of 1, 14, and chalcone (the
lead compound) that demonstrate their competitive behavior (effect
on Km only). Safinamide is shown as a
standard competitive inhibitor. As these compounds have different
inhibitory potencies, different concentrations were used to show the
effect on MAO-B.</p><p>The values of the inhibition
constant (Ki) for 1, 2, 13, 14, and 17 were
determined for both MAO-A and MAO-B and
are reported in Table 2 in a comparative analysis with the standard inhibitors safinamide
and isatin. From the data in Table 2, it clearly appears that the presence of the −CF3 group as the R1 substituent (at the meta (13) or para (14) position
of the phenyl ring) significantly improves both the potency of the
inhibition and the selectivity toward the MAO-B isoform with respect
to unsubstituted chalcone. Otherwise, the insertion of the −NO2 group as the R2 substituent at the meta position (1) is detrimental to both MAO-B inhibition
and the selectivity index (S.I.).</p><p>Values are reported as mean ±
SD of three independent experiments.</p><p>Selectivity index, given by S.I.
= Ki(MAO-A)/Ki(MAO-B).</p><p>See refs (19) and (23).</p><p>Standard MAO-B inhibitor.</p><p>Standard MAO-A and MAO-B inhibitor.</p><p>In particular, compound 13 emerges as the most potent
and selective inhibitor for MAO-B. Indeed, its inhibition constant
value (Ki = 5.0 nM) is lower than that
obtained for safinamide (Ki = 17 nM),
and it shows very good selectivity (S.I. = 920 vs 4820 of safinamide).
Finally, derivative 17 is less potent (Ki = 22 nM) and less selective (S.I. = 594) than 13 under our experimental conditions. It is worth mentioning
that the Ki value reported in Table 2 for 17 is lower than that previously reported in the literature19,23 because it was determined under different experimental buffer and
temperature conditions. In this regard, the 15–17 subseries was also tested previously on MAO-B, and for
both 15 and 16 a lower inhibitory potency
than for 17 was found.19,23</p><p>The
noteworthy inhibitory potency (Ki value
in the nanomolar range) and the high selectivity exerted by 13 point to this derivative as one of the most promising chalcone
scaffolds for the development of agents for neurodegenerative disease.
Indeed, comparable effects on MAO-B have been demonstrated only in
a few trisubstituted18 and disubstituted13 chalcone derivatives and in a furan-based chalcone
containing a cinnamyl group.17 The relevance
of 13 is further supported by the absence of cytotoxicity
also on nontumorigenic Met-5A mesothelial cells, for which a GI50 value higher than 20 Î¼M was obtained, as for safinamide.
In contrast, for the chalcone scaffold and 17, cytotoxicity
toward Met-5A was also confirmed, with GI50 values of about
7.0 ± 1.5 and 11.8 ± 1.1 μM, respectively.</p><p>Overall
these results prompted us to perform molecular modeling
studies on the most active compound of the series, i.e., 13.</p><p>First, in silico ADME predictions
made with QikProp
predicted that 13 has favorable drug-like properties
and good blood–brain barrier permeability (see Table S1). Then, docking calculations into representative
conformations of the human MAO-A and MAO-B proteins were performed
(see the Supporting Information for the
experimental details).24,26 In particular, two different
binding modes between 13 and MAO-B were predicted (termed
binding modes a and b), while a
single binding mode was observed for MAO-A. Figure 2a shows the binding mode of 13 in MAO-A, and Figure 2b depicts the more stable binding mode of 13 in MAO-B
(herein termed binding mode b). The less stable binding
mode of 13 in MAO-B (binding mode a)
is shown in Figure S1. The two binding
modes in MAO-B are likely due to the peculiar morphology of this isoform,
which is composed of two similar subcavities separated by the Ile199
and Tyr326 side chains.27,28 The binding modes are
similar to those previously reported for other chalcone inhibitors.21,29</p><p>Predicted
docking poses of 13 in the MAO-A and MAO-B
crystal structures: (a) binding mode of 13 in the MAO-A
protein (PDB ID 2Z5X); (b) the more energetically favored binding mode (binding mode b) predicted for 13 in the MAO-B crystal structure
(PDB ID 2V5Z).</p><p>The docking scores in MAO-B were
−10 kcal·mol–1 for binding mode a and −11 kcal·mol–1 for binding
mode b. The docking
score in MAO-A was −7.5 kcal/mol. To better discriminate between
the two binding modes in MAO-B, a rescoring of the predicted docking
poses was performed with a more rigorous free-energy-based screening
methodology (i.e., BEAR) to evaluate the binding
free energy of the ligand.30</p><p>According
to the binding free energy scores (ΔGbind) predicted by BEAR, binding mode b of 13 turned out to be 6.8 kcal·mol–1 more stable
than binding mode a (Table S2). In binding mode b, the B phenyl
ring of 13 establishes hydrophobic interactions with
the aromatic rings of the Tyr398, Tyr435, and Tyr60 side chains and
the flavine ring of FAD. The α,β-unsaturated ketone binds
close to the Ile198 and Leu171 side chains, with the carbonyl moiety
forming a hydrogen bond with the Cys172 side chain, as previously
observed for other chalcone compounds.29 The A ring forms hydrophobic contacts with the Leu164, Leu167, Phe168
and Trp119 side chains, as previously observed in ref (29). In particular, the trifluoromethyl
moiety was predicted to be accommodated near the Trp119 and Leu164
side chains, establishing close contacts. The hydrogen bond of the
carbonyl with the Cys172 residue is of special interest because this
residue, which is mutated to Asn181 in MAO-A, has a recognized role
in MAO-B/MAO-A selectivity.21,29 Importantly, docking
of 13 into MAO-A did not provide an orientation similar
to that of MAO-B. In MAO-A, the trifluoromethylbenzene moiety (ring
A) was predicted to be accommodated toward the entrance of the MAO-A
binding site near residues Leu97, Ile335, and Leu337 because of steric
clashes with the Phe208 side chain. Moreover, the carbonyl group of 13 did not establish hydrogen bonds with MAO-A residues. Interestingly,
the lack of a hydrogen bond with residues in this region of the MAO-A
binding pocket, like those previously observed with Cys172 or Tyr326
into MAO-B, has been reported to play an important role in isoform
selectivity for the MAO proteins.27,28,31 The B phenyl ring is accommodated close to the Tyr60
and Tyr407 side chains and FAD, similar to what was observed in MAO-B.
Altogether, these differences help explain the experimentally observed
selectivity of compound 13 for MAO-B over MAO-A.</p><p>Considering that docking calculations on 13 suggested
two alternative orientations, one of which was clearly favored over
the other, we sought to investigate whether para (compound 14) instead of meta (compound 13) trifluorocarbon substitution could play a role in discriminating
the ligand orientation. To this aim, the binding modes of 13 and 14 in MAO-B were studied by X-ray crystallography.
The three-dimensional structures of human MAO-B in complex with 13 (Figures 3 and 4a) and 14 (Figure 4b) were solved at 2.3 and 2.1
Å resolution, respectively (statistics are reported in Table S3). For both structures, the overall fold
and, in particular, the active site and the bound inhibitor display
the same conformation between the two monomers of the dimer contained
in the asymmetric unit (the root-mean-square deviations for CÎ±
atoms are 0.30 and 0.27 Å between chains A and B for the structures
in complex with compounds 13 and 14, respectively).
Hereafter, we will refer to subunit A of each structure for the following
discussion.</p><p>Overall structure of the MAO-B dimer represented as a pink ribbon
diagram (chain A is on the left) with the membrane-spanning C-terminal
helix pointing to the bottom of the figure. The FAD cofactor is shown
in stick representation with carbon, nitrogen, oxygen, and phosphorus
atoms colored in yellow, blue, red, and magenta, respectively. The
structure in complex with 13 (in green, with fluorine
atoms in light blue and oxygen in red) is shown. The overall fold
is identical to that of the enzyme in complex with 14.</p><p>Architectures of the MAO-B active site in complex
with (a) 13 and (b) 14. FAD is depicted
as in Figure 3. The
residues that
line the cavity are represented as sticks with carbon, nitrogen, oxygen,
and sulfur atoms in pink, blue, red, and orange, respectively. The
refined 2Fo – Fc electron density map for the inhibitor molecule (contoured
at 1.2σ) is shown in blue chicken-wire style. The fluorine atoms
of both inhibitors are in light blue, with carbon atoms in green (13) and gray (14). Water molecules are depicted
as red spheres and hydrogen bonds as dashed lines.</p><p>Inspection of the electron density in the enzyme active site
revealed
that 13 and 14 bind in the hydrophobic cavity
of MAO-B (Figure 4a,b,
respectively). In both cases the chalcone moiety is accommodated with
the carbonyl oxygen pointing toward the bottom of the active site
and establishing a hydrogen bond with Cys172 in one of the two conformations
that this residue adopts. This feature was also found in the MAO-B
structures in complex with chromone inhibitors.32 The distances between the cysteine side chain and the chalcone
alkene unit are 4.0 and 4.6 Å for 13 and 14, respectively, both of which are too long for the formation of a
covalent bond as previously observed for other targets.33 Compound 13 binds with the CF3 substituent in the entrance cavity space (Figure 4a), which is in agreement with
the most stable orientation predicted by modeling as described above
and is consistent with previous studies highlighting this part of
the active site as a favorable niche for halogen atoms (such as in
the case of safinamide; see Figure 5). Most likely, in the crystallized MAO-B–13 complex the predicted binding mode b of 13 is selected. Interestingly, 14 is bound in
the opposite way with respect to the flavin compared with 13, with the CF3 group positioned within the aromatic cage
formed by residues Tyr398 and Tyr435 in front of the flavin (Figure 4b). This is unique
in MAO-B structures and is probably due to the para position of the substituent on the aromatic ring, which would clash
with the residues belonging to the gating loop formed by residues
99–110 (on the left in Figure 4). Similar to the fluorine atom substituent in safinamide,
the CF3 group of 13 is at the meta position and can easily be accommodated in the entrance cavity.
Superposition of the MAO-B structures in complex with the two chalcone
inhibitors highlights that the carbonyl–Cys172 hydrogen bond
functions as a sort of pivot point by fixing the molecules in the
active site and orienting them in opposite directions with respect
to the flavin (Figure 5). The chalcone scaffold is constrained by the flat and rigid MAO-B
cavity to position the inner aromatic ring perpendicular to the flavin
and the other slightly tilted, similar to the conformation observed
in safinamide (Figure 5). Retrospective docking calculations predicted only one binding
mode for 14 (docking score of −10 kcal·mol–1), consistent with the orientation observed in the
crystal structure and similar to the predicted binding mode a of 13 (Figure S2).</p><p>Superposition of MAO-B structures in complex with 13, 14, and safinamide (the latter represented with carbon
atoms in magenta; PDB code 2V5Z).24 For the sake of clarity,
water molecules and hydrogen bonds shown in Figure 4 have been omitted.</p><p>In conclusion, we report that among a library of monosubstituted
structurally related chalcones, three derivatives (1, 13, and 14) demonstrated a safe profile on different
human cell lines and then were considered for further investigation.
In particular, 13 appeared to be good candidate to target
MAO enzymes in the context of neurodegenerative diseases. Indeed,
it behaves as competitive reversible inhibitor of MAO-B, showing an
inhibitory potency of Ki = 5 nM (which
is higher than that of the clinically used safinamide) and a remarkable
selectivity toward the MAO-B isoform. Docking studies of the binding
of 13 to MAOs confirmed the strong affinity and selectivity
for MAO-B versus MAO-A by predicting opposite orientations in the
active site of the two isoforms (with the trifluoromethylbenzene moiety
pointing toward the FAD cofactor in MAO-B and toward the active-site
entrance in MAO-A). Crystal structures of MAO-B in complex with 13 and 14 highlighted the key role played by
the position of the trifluoromethyl moiety on the phenyl ring (meta in the former and para in the latter)
in orienting the inhibitor into the active site.</p><p>Overall, our
studies pointed to 13 as a very promising
chalcone derivative to be considered for the development of novel
MAO-B-selective inhibitors for the treatment of neurodegenerative
diseases. The evaluation of its effectiveness and nontoxicity in vivo and the effect on other targets involved in neurodegenerative
disorders warrant further investigation.</p><p>Chemistry, materials
and methods, molecular docking,
crystallographic details, and NMR spectra (PDF)</p><p>ml1c00238_si_001.pdf</p><!><p>∇ L.G.I.,
L.P., and G.F. contributed equally</p><!><p>The authors declare no
competing financial interest.</p>