Due to the neuroprotective role of the Na+/Ca2+ exchanger (NCX) isoforms NCX1 and NCX3, we synthesized novel benzodiazepinone derivatives of the unique NCX activator Neurounina-1, named compounds 1-19. The derivatives are characterized by a benzodiazepinonic nucleus linked to five- or six-membered cyclic amines via a methylene, ethylene, or acetyl spacer. The compounds have been screened on NCX1/NCX3 isoform activities by a high-throughput screening approach, and the most promising were characterized by patch-clamp electrophysiology and Fura-2AM video imaging. We identified two novel modulators of NCX: compound 4, inhibiting NCX1 reverse mode, and compound 14, enhancing NCX1 and NCX3 activity. Compound 1 displayed neuroprotection in two preclinical models of brain ischemia. The analysis of the conformational and steric features led to the identification of the molecular volume required for selective NCX1 activation for mixed NCX1/NCX3 activation or for NCX1 inhibition, providing the first prototypal model for the design of optimized isoform modulators.
Due to the neuroprotective role of the Na+/Ca2+ exchanger (NCX) isoforms NCX1 and NCX3, we synthesized novel benzodiazepinone derivatives of the unique NCX activator Neurounina-1, named compounds 1-19. The derivatives are characterized by a benzodiazepinonic nucleus linked to five- or six-membered cyclic amines via a methylene, ethylene, or acetyl spacer. The compounds have been screened on NCX1/NCX3 isoform activities by a high-throughput screening approach, and the most promising were characterized by patch-clamp electrophysiology and Fura-2AM video imaging. We identified two novel modulators of NCX: compound 4, inhibiting NCX1 reverse mode, and compound 14, enhancing NCX1 and NCX3 activity. Compound 1 displayed neuroprotection in two preclinical models of brain ischemia. The analysis of the conformational and steric features led to the identification of the molecular volume required for selective NCX1 activation for mixed NCX1/NCX3 activation or for NCX1 inhibition, providing the first prototypal model for the design of optimized isoform modulators.
Calcium
(Ca2+) ions play an important role in the control
of virtually all cellular functions. Maintenance of Ca2+ homeostasis is due to the concerted activity of several proteins
expressed not only on plasma membrane but also in intracellular organelles
storing Ca2+.[1] Among the proteins
handling Ca2+, the Na+/Ca2+ exchanger
(NCX) has attracted much attention for having, among other electrophysiological
features, a high transport capacity. This is of particular importance
in those conditions characterized by elevated Ca2+ levels
such as during the occurrence of neurodegeneration associated to neurological
diseases[2,3] and during myocardial ischemia–reperfusion
injury.[4]Na+/Ca2+ exchangers belong to a superfamily
of Ca2+/cation antiporters.[5] Three isoforms named NCX1, NCX2, and NCX3 have been identified and
characterized.[6,7] These proteins display different
localizations: NCX1 is widely expressed in various organs and tissues,
whereas NCX2 and NCX3 show a more restricted expression. It is worthy
accredited that two of these isoforms, NCX1 and NCX3, are localized
intracellularly in specific organelles, where they play an important
role in handling the local Ca2+ level.[8,9] Of
note, calcium handling outside the mitochondria requires two consecutive
steps involving the operation of the Na+-sensitive NCLX
which may transport either Li+ or Na+ in exchange
for Ca2+,[10] mediating Ca2+ transport from the matrix to the intermembrane space, and
mNCX3, promoting Ca2+ efflux from the intermembrane space
to the cytosol.[9]From the functional
point of view, Ca2+ and Na+ ions are transported
by the exchanger through the occupancy of their
transport sites. However, they also exert a modulatory role on NCX
activity. Accordingly, the exchanger activity is regulated in an allosteric
way by the binding of Ca2+ and Na+ ions to specific
NCX domains. For instance, Ca2+ binding at the cytosolic
face of NCX1 does activate the exchanger, while Na+ binding
at the cytosolic face exerts inhibitory modulation.[11−14]In the last 30 years, the
occurrence of [Na+] and [Ca2+] dyshomeostasis has been reported
in several neurodegenerative diseases
either at neuronal or glial components. Altered expression and activity
of NCX isoforms have been also demonstrated in stroke, multiple sclerosis
(MS), amyotrophic lateral sclerosis, SMA, Parkinson’s disease,
and Alzheimer’s disease.[15−21] Although the role of each isoform is still under examination, many
lines of evidence point toward a neuroprotective effect of NCX activation
in most of these neurodegenerative diseases.[21]Concerning the molecular mechanism underlying the neuroprotection
exerted by NCX1 in stroke pathogenesis, functional studies demonstrated
its ability to refill the endoplasmic reticulum with Ca2+ ions when it works in the reverse mode during hypoxic conditions
and ischemic preconditioning (PC).[22,23]This
mechanism is also shared by the NCX3 isoform whose reverse
mode may contribute to delay ER stress in hippocampal neurons exposed
to Aβ1-42.[20,24]Neurounina-1 (Figure ) is an
activator of both the reverse and forward mode
of NCX1 and NCX2 and it was validated in preclinical models of stroke
as a neuroprotective agent.[25] Then, very
recently, its pharmacokinetic profile has been screened in vivo.[26]
Figure 1
Chemical structure of Neurounina-1.
Chemical structure of Neurounina-1.In the present study, we synthesized and characterized
novel benzodiazepinonic
derivatives of Neurounina-1 (compounds 1–19; Table ) with the
aim to provide new insights into the structure–activity relationship
(SAR) of NCX isoform modulators. All these compounds have been screened
with several approaches moving from functional high-throughput screening
to patch-clamp electrophysiology and Fura-2AM video-imaging on single-cells.
Furthermore, the most selective derivatives have been tested in two
different in vitro models of brain ischemia on primary cortical neurons.
Computational studies combined with experimental data allowed us to
produce the first prototypal model for the design of new molecular
entities acting on NCX proteins.
Table 1
Chemical Structures
of Compounds 1–19
Results and Discussion
Design and Synthesis of Neurounina-1 Derivatives
(Compounds 1–19)
The chemical structures
of compounds 1–19 are reported in Table . The synthetic strategy for
the preparation of compounds 1–3 is reported in Scheme ; compounds were
obtained following, with modifications, the procedures previously
reported for the synthesis of Neurounina-1.[25]
Scheme 1
Reagents and Conditions: (a) Formaldehyde
Solution 37% wt in H2O (10 equiv), Pyrrolidine (Compounds 1 and 2; 10 equiv) or Piperidine (Compound 3; 10 equiv),
and Glacial Acetic Acid, MW (500 W, 15 min; T = 80
°C)
The strategies for the synthesis
of the compounds 4–11 and 12–19 are summarized in Schemes and 3, respectively. Compounds 4–11 (Scheme ) were obtained by the nucleophilic
substitution of nitrazepam with 1-bromo-2-chloroethane in the presence
of NaH (60% dispersion in mineral oil) in dimethylformamide (DMF)
that led to intermediate A that was subsequently condensed
with the selected cyclic amines (ACy, i–viii)
in acetonitrile in the presence of K2CO3 and
NaI.
Scheme 2
Reagents and Conditions: (a) NaH (60% Dispersion in Mineral
Oil),
0 °C, DMF then 4 h, rt; (b) ACy (i–viii),
K2CO3 (1.1 equiv), NaI (1.1 equiv), CH3CN, Reflux, 3 h
Scheme 3
Reagents and Conditions:
(a) NaH (60% Dispersion in Mineral Oil),
0 °C, DMF and Then 4 h, rt; (b) Anhydrous CH2Cl2/TFA (8:2, v/v, 20 mL), rt, 1 h; (c) ACy (i–iv and vii–viii), TBTU (1.1 equiv), HOBt (1.1 equiv),
DIPEA, DMF, Overnight, rt; and (d) (1) SOCl2 (10 equiv),
Anhydrous CH2Cl2, Reflux, 1 h and (2) ACy (v and vi, 2 equiv), Anhydrous Tetrahydrofuran,
Overnight, rt
The general procedure
for the synthesis of compounds 12–19 (Scheme ) is as
follows: alkylation of nitrazepam with tert-butyl
bromoacetate in the presence of NaH (60% dispersion in mineral oil)
in anhydrous DMF gave intermediate B. The reaction with
trifluoroacetic acid in anhydrous dichloromethane (20% v/v) allowed
the preparation of the carboxylic intermediate C that
was successively coupled with the opportune cyclic amines (i–iv and vii–viii) in the presence of TBTU, HOBt,
and diisopropylethylamine in DMF to give the desired compounds 12–15 and 18–19. For the preparation
of 16 and 17, intermediate C was converted into the corresponding acyl chloride by reaction with
thionyl chloride in anhydrous dichloromethane and was further coupled
with imidazole (v) and 1-methyl-imidazole (vi) in a 1:2 M ratio at room temperature in anhydrous tetrahydrofuran.
Molecular Characterization of the Newly Synthesized Compounds
on NCX1 and NCX3 Activity
In order to identify new compounds
able to modulate NCX activity in the reverse mode, BHK-NCX1 and BHK-NCX3
cells were loaded with Fluo-4 (Figure ) or Fura-2 (Figure S1)
and then superfused in the high-throughput screening setting with
a single pulse of Na+-deficient NMDG+ medium
(Na+-free) in the presence of 10 nM and 10 μM concentrations
of each compound in the case of Fluo-4 experiments or in the presence
of 10 μM of each compound in the case of Fura2-loaded cells.
Among all the 19 compounds screened, we identified a selective NCX1
inhibitor, compound 4, and two activators, compounds 1 and 14. In particular, compound 14 was able to stimulate NCX1 and NCX3, whereas compound 1 resulted in a selective NCX1 activator, confirming previous results
obtained by single-cell Fura-2-monitored Ca2+ influx techniques.[27]
Figure 2
High-throughput screening of the novel benzodiazepinone
derivative
efficacy on NCX activity in the reverse mode in BHK-NCX1 and BHK-NCX3
cells loaded by Fluo-4. (A) Effect of compound 14 (10
nM and 10 μM), compound 1 (10 nM and 10 μM),
and Neurounina-1 (10 nM) on NCX1 in BHK-NCX1 cells. *p < 0.05 vs control. (B) Effect of the same compounds
on NCX3 in BHK-NCX3 cells. *p < 0.05 vs control, Neurounina-1, and compound 1. (C) Effect of two
different concentrations of compound 4 on the NCX1 reverse
mode in BHK-NCX1 cells. *p < 0.05 vs Neurounina-1 and internal control (untreated cells). The effects of compounds 1–19 on NCX1 and NCX3 reverse mode activity measured
in BHK-NCX1 and BHK-NCX3 loaded by Fura-2 AM have been reported in Figure S1.
High-throughput screening of the novel benzodiazepinone
derivative
efficacy on NCX activity in the reverse mode in BHK-NCX1 and BHK-NCX3
cells loaded by Fluo-4. (A) Effect of compound 14 (10
nM and 10 μM), compound 1 (10 nM and 10 μM),
and Neurounina-1 (10 nM) on NCX1 in BHK-NCX1 cells. *p < 0.05 vs control. (B) Effect of the same compounds
on NCX3 in BHK-NCX3 cells. *p < 0.05 vs control, Neurounina-1, and compound 1. (C) Effect of two
different concentrations of compound 4 on the NCX1 reverse
mode in BHK-NCX1 cells. *p < 0.05 vs Neurounina-1 and internal control (untreated cells). The effects of compounds 1–19 on NCX1 and NCX3 reverse mode activity measured
in BHK-NCX1 and BHK-NCX3 loaded by Fura-2 AM have been reported in Figure S1.Interestingly, compound 1 activity has been recently
linked to learning and memory function through the NCX1-dependent
CaMKIIα phosphorylation.[24] Furthermore,
the involvement of NCX3 isoform in hippocampal long-term potentiation
and spatial learning and memory has also been demonstrated.[28] Therefore, the newly synthesized compound 14 was selected to compare its putative neuroprotective profile
with those of compound 1 under anoxic/hypoxic conditions.
In this respect, we adopted two different anoxic/hypoxic models consisting
on the exposure of primary cortical neurons to oxygen and glucose
deprivation (OGD), followed by reoxygenation (RX) or chemical hypoxia
followed by RX (see the next paragraph).Furthermore, NCX activity
in the reverse mode of operation was
also studied on single cell by Fura-2AM video-imaging in BHK-NCX1
and BHK-NCX3 cells (Figures and 4). In these stably transfected
cells, Na+-free perfusion caused a rapid rise in [Ca2+], which was significantly enhanced,
in a dose-dependent way, by compound 14 (EC50 = 3.5 nM for NCX1 and EC50 = 2 μM for NCX3; Figures and 4). Furthermore, patch-clamp electrophysiology in the whole-cell
configuration was used to measure NCX activity both in the forward
and reverse mode of operation.
Figure 3
Effect of compound 14 on
NCX1 activity in BHK-NCX1
cells. (A,B) Representative superimposed traces and quantification
of the effect of compound 14 on Na+-free-induced
[Ca2+] increase through NCX1
reverse mode of operation in Fura-2-loaded BHK-NCX1 cells. The bar
graph reports the mean ± SEM of the maximal [Ca2+] responses measured in approximately 60 cells
per group. Averaged data from four different experimental sessions
were normalized as the percentage of controls. *p < 0.05 vs control and 1 nM; **p < 0.05 vs
all. (C,D) Representative INCX traces recorded by whole-cell
patch-clamp electrophysiology in control cells and in cells treated
with compound 14 (10 nM). The bar graphs in (D) report
the mean ± SEM of the forward and reverse NCX1 current densities,
respectively, measured in at least 10 cells for each experimental
group. Reverse INCX amplitude was measured at +60 mV, while
forward INCX was measured at −120 mV. *p < 0.05 vs each internal control.
Figure 4
Effect
of compound 14 on NCX3 activity in BHK-NCX3
cells. (A,B) Representative superimposed traces and quantification
of the effect of compound 14 on Na+-free-induced
[Ca2+] increase through NCX3
reverse mode of operation in Fura-2-loaded BHK-NCX3 cells. The bar
graph reports the mean ± SEM of the maximal [Ca2+] responses measured in approximately 50 cells
per group. Averaged data from four different experimental sessions
were normalized as the percentage of controls. *p < 0.05 vs control and 1 μM; **p < 0.05
vs all. (C,D) Representative INCX traces recorded by whole-cell
patch-clamp electrophysiology in control cells and in cells treated
with compound 14 (10 μM). The bar graphs in (D)
report the mean ± SEM of the forward and reverse NCX3 current
densities, respectively, measured in nine cells for each experimental
group. Reverse INCX amplitude was measured at +60 mV, while
forward INCX was measured at −120 mV. *p < 0.05 vs each internal control.
Effect of compound 14 on
NCX1 activity in BHK-NCX1
cells. (A,B) Representative superimposed traces and quantification
of the effect of compound 14 on Na+-free-induced
[Ca2+] increase through NCX1
reverse mode of operation in Fura-2-loaded BHK-NCX1 cells. The bar
graph reports the mean ± SEM of the maximal [Ca2+] responses measured in approximately 60 cells
per group. Averaged data from four different experimental sessions
were normalized as the percentage of controls. *p < 0.05 vs control and 1 nM; **p < 0.05 vs
all. (C,D) Representative INCX traces recorded by whole-cell
patch-clamp electrophysiology in control cells and in cells treated
with compound 14 (10 nM). The bar graphs in (D) report
the mean ± SEM of the forward and reverse NCX1 current densities,
respectively, measured in at least 10 cells for each experimental
group. Reverse INCX amplitude was measured at +60 mV, while
forward INCX was measured at −120 mV. *p < 0.05 vs each internal control.Effect
of compound 14 on NCX3 activity in BHK-NCX3
cells. (A,B) Representative superimposed traces and quantification
of the effect of compound 14 on Na+-free-induced
[Ca2+] increase through NCX3
reverse mode of operation in Fura-2-loaded BHK-NCX3 cells. The bar
graph reports the mean ± SEM of the maximal [Ca2+] responses measured in approximately 50 cells
per group. Averaged data from four different experimental sessions
were normalized as the percentage of controls. *p < 0.05 vs control and 1 μM; **p < 0.05
vs all. (C,D) Representative INCX traces recorded by whole-cell
patch-clamp electrophysiology in control cells and in cells treated
with compound 14 (10 μM). The bar graphs in (D)
report the mean ± SEM of the forward and reverse NCX3 current
densities, respectively, measured in nine cells for each experimental
group. Reverse INCX amplitude was measured at +60 mV, while
forward INCX was measured at −120 mV. *p < 0.05 vs each internal control.The whole-cell current was measured at +60 mV (reverse mode) and
−120 mV (forward mode) using a ramp-clamp protocol for NCX
currents (see Methods). To isolate NCX currents (INCX),
cells were recorded for 5 min with the well-known NCX inhibitor NiCl2 (5 mM). The Ni2+-sensitive component, representing
the isolated INCX, was obtained as previously reported.[15,29] No current corresponding to INCX was recorded in BHK-Wt
cells.[29] Besides its highest potency in
modulating NCX1, compound 14 displayed a greater selectivity
for the NCX3 forward mode.Accordingly, while compound 14 strongly enhanced both
the outward (reverse mode) and inward (forward mode) direction of
INCX1 and INCX3 (Figures and 4), it showed
a much higher effect (i) on the INCX1 reverse mode than
on its forward mode and (ii) on the INCX3 forward mode
than on its reverse mode.Moreover, compound 4 displayed
specific inhibitory
action on NCX1, thus representing a new selective inhibitor of the
exchanger isoform 1 (Figure C). Accordingly, in BHK-NCX1 cells, compound 4 reduced, in a concentration-dependent manner (IC50 =
10 μM), Na+-free induced [Ca2+] increase mediated by the exchanger isoform reverse
mode. Interestingly, compound 4 was more efficacious
on the reverse mode of NCX1 than on its forward mode as measured by
patch-clamp electrophysiology in the whole-cell configuration (Figure A,C). In contrast,
it failed to modulate the NCX activity in BHK-NCX3 cells (Figures B and S1).
Figure 5
Effect of compound 4 on NCX1 and
NCX3 activity in
BHK-NCX1 and BHK-NCX3 cells. (A) Representative INCX traces
recorded in BHK-NCX1 by whole-cell patch-clamp electrophysiology in
control cells and in cells treated with compound 4 (10
μM). The bar graphs in (A) report the mean ± SEM of the
reverse and forward NCX1 current densities, respectively, measured
in 10 cells for each experimental group. Reverse INCX amplitude
was measured at +60 mV, while forward INCX was measured
at −120 mV. *p < 0.05 vs each internal
control. (B) Representative INCX traces recorded in BHK-NCX3
by whole-cell patch-clamp electrophysiology in control cells and in
cells treated with compound 4 (10 μM). The bar
graphs in (B) report the mean ± SEM of the reverse and forward
NCX1 current densities, respectively, measured in 10 cells for each
experimental group. Reverse INCX amplitude was measured
at +60 mV, while forward INCX was measured at −120
mV. (C) Quantification of the concentration-dependent effect of compound 4 on Na+-free-induced [Ca2+] increase through NCX1 reverse mode of operation
in Fura-2-loaded BHK-NCX1 cells. The bar graph reports the mean ±
SEM of the maximal [Ca2+] responses
measured in approximately 30 cells per group. Averaged data from four
different experimental sessions were normalized as the percentage
of controls. *p < 0.05 vs control, 1, and 1 0
nM; **p < 0.05 vs all.
Effect of compound 4 on NCX1 and
NCX3 activity in
BHK-NCX1 and BHK-NCX3 cells. (A) Representative INCX traces
recorded in BHK-NCX1 by whole-cell patch-clamp electrophysiology in
control cells and in cells treated with compound 4 (10
μM). The bar graphs in (A) report the mean ± SEM of the
reverse and forward NCX1 current densities, respectively, measured
in 10 cells for each experimental group. Reverse INCX amplitude
was measured at +60 mV, while forward INCX was measured
at −120 mV. *p < 0.05 vs each internal
control. (B) Representative INCX traces recorded in BHK-NCX3
by whole-cell patch-clamp electrophysiology in control cells and in
cells treated with compound 4 (10 μM). The bar
graphs in (B) report the mean ± SEM of the reverse and forward
NCX1 current densities, respectively, measured in 10 cells for each
experimental group. Reverse INCX amplitude was measured
at +60 mV, while forward INCX was measured at −120
mV. (C) Quantification of the concentration-dependent effect of compound 4 on Na+-free-induced [Ca2+] increase through NCX1 reverse mode of operation
in Fura-2-loaded BHK-NCX1 cells. The bar graph reports the mean ±
SEM of the maximal [Ca2+] responses
measured in approximately 30 cells per group. Averaged data from four
different experimental sessions were normalized as the percentage
of controls. *p < 0.05 vs control, 1, and 1 0
nM; **p < 0.05 vs all.
Effect of the Benzodiazepinone Derivatives, Compounds 1 and 14, Enhancing NCX Activity, and Compound 4, Inhibiting NCX Activity, on Cell Survival of Primary Cortical
Neurons Exposed to Hypoxic Conditions Mimicking Stroke
Once
identified as NCX activity modulators, compounds 1, 4, and 14 were tested on cell viability of primary
rat cortical neurons exposed to OGD followed by RX, an experimental
condition mimicking hypoxic conditions occurring during stroke. These
compounds were added during either OGD or RX phases at the corresponding
median effective concentration for their NCX stimulating or inhibiting
activity.Under these anoxic conditions, only compound 1 showed a neuroprotective profile in cortical neurons (Figure A) in a concentration-dependent
way (Figure S2). Furthermore, when compound 1 was preincubated in rat primary cortical neurons and then
removed during the exposure to OGD and RX phases, it continued to
protect neurons from hypoxic conditions (Figure B). Considering that this represents a more
demanding experimental paradigm, compound 1 may be considered
an interesting tool with a neuroprotective profile. In fact, its neuroprotective
effect in cortical neurons exposed to hypoxic conditions was similar
to that produced by ischemic PC,[30,31] a short stimulus
inducing ischemic tolerance in neurons (Figure B). Furthermore, to better investigate the
neuroprotective profile of compound 1, it was tested in primary cortical
neurons exposed to chemical hypoxia, followed by RX, a detrimental
condition undermining the mitochondrial function. Under these conditions,
compound 1 significantly reduced cell suffering (Figure C) in a concentration-dependent
way (Figure S2), thus reinforcing the concept
of its neuroprotective profile under anoxic/hypoxic conditions. Interestingly,
NCX1 and NCX3 are upregulated in those brain regions protected by
PC treatment.[30,31] However, the specific role of
each mode of operation in the neuroprotective effect remains obscure
and very difficult to define in this experimental paradigm. In consideration
of our data showing the unique neuroprotective effect of compound 1 and the lack of neuroprotection by compound 14, we could speculate that in general, the ability to stimulate the
NCX reverse mode could have a major readout than that of forward mode
stimulation. In this view, the much greater effect of compound 14 on the forward mode of NCX3 than on its reverse mode could
also play a role. Indeed, previous studies showed a neuroprotective
effect of ER Ca2+ refilling mediated by NCX both during
persistent anoxic conditions and ischemic PC.[22,23] Therefore, the excessive activation of the NCX3 forward mode determining
Ca2+ extrusion may negatively counterbalance the activation
of its reverse mode, favoring Ca2+ entrance.
Figure 6
Neuroprotective
effect of compound 1 in primary cortical
neurons exposed to OGD followed by RX or chemical hypoxia followed
by RX. (A) Bar graph depicting the effect on cell viability of compounds 1 (10 nM), 14 (10 μM), and 4 (10 μM) added during both OGD and RX. Cell viability was measured
at the end of RX. Data are means ± SEM of five separate experiments
and are reported as the percentage of cell viability in the control
(100% viability). *p < 0.05 vs normoxic cells
(control); **p < 0.05 vs OGD/Rx. (B) Bar graph
depicting the effect on cell viability of compound 1 preincubated
before OGD + RX. After preincubation, compound 1 was
removed during OGD + RX and it was compared to ischemic PC. *p < 0.05 vs normoxic cells (control); **p < 0.05 vs OGD/Rx. (C) Bar graph depicting the effect on cell
viability of compound 1 (10 nM) added during both chemical
hypoxia (Ch Hyp, 45′) and RX (3 h). Cell viability was measured
at the end of RX. Data are means ± SEM of three separate experiments
and are reported as the percentage of cell viability in the control
(100% viability). *p < 0.05 vs normoxic cells
(control); **p < 0.05 vs Ch Hyp + Rx.
Neuroprotective
effect of compound 1 in primary cortical
neurons exposed to OGD followed by RX or chemical hypoxia followed
by RX. (A) Bar graph depicting the effect on cell viability of compounds 1 (10 nM), 14 (10 μM), and 4 (10 μM) added during both OGD and RX. Cell viability was measured
at the end of RX. Data are means ± SEM of five separate experiments
and are reported as the percentage of cell viability in the control
(100% viability). *p < 0.05 vs normoxic cells
(control); **p < 0.05 vs OGD/Rx. (B) Bar graph
depicting the effect on cell viability of compound 1 preincubated
before OGD + RX. After preincubation, compound 1 was
removed during OGD + RX and it was compared to ischemic PC. *p < 0.05 vs normoxic cells (control); **p < 0.05 vs OGD/Rx. (C) Bar graph depicting the effect on cell
viability of compound 1 (10 nM) added during both chemical
hypoxia (Ch Hyp, 45′) and RX (3 h). Cell viability was measured
at the end of RX. Data are means ± SEM of three separate experiments
and are reported as the percentage of cell viability in the control
(100% viability). *p < 0.05 vs normoxic cells
(control); **p < 0.05 vs Ch Hyp + Rx.
Computer-Aided SAR Analysis
In order to analyze SARs
of Neurounina-1 and compounds 1–19, computational studies have been performed. Molecular simulations
were carried out considering the compounds in their calculated prevalent
ionic format blood (7.4) and cytoplasmic (7.2) pH values (Table S1); accordingly, Neurounina-1 and compounds 1–7 were considered protonated,
while compounds 8–19 were considered neutral.
The conformational space of the compounds has been sampled by using
a stochastic conformational search algorithm, while the generalized
Born model has been applied to mimic an aqueous environment. Resulting
conformers were ranked by their conformational energy, analyzed, and
classified according to their conformational features (see the Experimental Section for details). The spatial orientation
of the R groups with respect to the benzodiazepinone ring was mapped,
and the conformers were accordingly classified into families (Tables S2–S21). This allowed the identification
of the most favored conformational family of each compound (Table and Figure ) and the generation of a ligand-based
pharmacophore model according to the available experimental data (Figure ; see the Experimental Section for details).
Table 2
Lowest Energy Conformational Families
of Neurounina-1 and Compounds 1–19a
torsional
anglesc,d (deg)
compound
family
ΔEGMb (kcal/mol)
occurrence rate (%)
τ1
τ2
Neurounina-1
Ic
0.00
11
–57
175
1
Ic
0.00–1.34
22
–55
177
2
Ic
0.00
14
–58
179
3
Ic
0.00
20
–64
173
4
IIc
0.00–0.92
33
74
–179
5
IIc
0.00–0.82
26
74
–175
6
IIc
0.00–3.0 2
15
74
–171
7
IIc
0.00–3.00
22
74
–175
8
Ib
0.00–0.11
32
–67
–59
9
Ib
0.00–0.01
36
–67
–55
10
Ib
0.00–4.77
42
–80
–61
11
Ib
0.00–4.56
57
–75
–63
12
IIIc
0.00–0.31
50
–133
–165
13
IIIc
0.00–0.35
57
–130
–168
14
IIIc
0.00–0.63
42
–130
–169
15
IIIc
0.00–0.56
31
–128
–166
16
IIIa
0.00–0.97
37
–143
59
17
IIIa
0.00
37
–143
60
18
IIIc
0.00–3.51
56
–127
–172
19
IIIc
0.00–4.97
59
–111
–145
Torsional angle, ΔEGM, and occurrence rate values are reported.
The values reported refer to the
lowest and the highest energy conformers of the family, the presence
of just one value means that all conformers converged to the same
structure.
The conformational/configurational
enantiomer with the opposite flip present the same absolute values
of all torsional angles with the opposite sign.
The values reported refer to the
lowest energy conformers of each family.
Figure 7
Lowest energy conformers
of Neurounina-1 and compounds 1–19 superimposed by the heavy atoms of the benzodiazepinone
ring system. (A) Compounds with aliphatic R groups: Neurounina-1, compounds 1–3, 4–7, and 12–15. (B) Compounds with an aromatic R group: 8–11 and 16–19. Carbon atoms are
colored according to the spacer group: Neurounina-1,
compounds 1–3 (carbon atoms: green), 4–11 (carbon atoms: orange), and 12–19 (carbon atoms:
magenta). The active analogues (Neurounina-1, compounds 1, 4, and 14) are displayed as ball
and sticks; the inactive analogues are displayed as sticks. Heteroatoms
are colored by atom type; hydrogen atoms are omitted for clarity of
presentation except for those involved in hydrogen bonds. Hydrogen
bonds are displayed as green dashed lines.
Lowest energy conformers
of Neurounina-1 and compounds 1–19 superimposed by the heavy atoms of the benzodiazepinone
ring system. (A) Compounds with aliphatic R groups: Neurounina-1, compounds 1–3, 4–7, and 12–15. (B) Compounds with an aromatic R group: 8–11 and 16–19. Carbon atoms are
colored according to the spacer group: Neurounina-1,
compounds 1–3 (carbon atoms: green), 4–11 (carbon atoms: orange), and 12–19 (carbon atoms:
magenta). The active analogues (Neurounina-1, compounds 1, 4, and 14) are displayed as ball
and sticks; the inactive analogues are displayed as sticks. Heteroatoms
are colored by atom type; hydrogen atoms are omitted for clarity of
presentation except for those involved in hydrogen bonds. Hydrogen
bonds are displayed as green dashed lines.Torsional angle, ΔEGM, and occurrence rate values are reported.The values reported refer to the
lowest and the highest energy conformers of the family, the presence
of just one value means that all conformers converged to the same
structure.The conformational/configurational
enantiomer with the opposite flip present the same absolute values
of all torsional angles with the opposite sign.The values reported refer to the
lowest energy conformers of each family.All compounds were characterized by the presence of
two possible
flips of the benzodiazepinone system, which produced two sets of isoenergetic
conformers (named A and B) with nonsuperimposable, specular structures
(conformational enantiomers; Figure S3);
in the case of the chiral derivatives 6, 7, 14, and 15, tested as racemic mixtures,
the specular conformers corresponded to those of the two configurational
enantiomers. The torsion angle values in Table are referred to the A set of conformers
(τflip = −65°; S-chirality), and the
B set of conformers (τflip = 65°; R-chirality)
presented the same energy values and the torsion angle values with
the opposite sign. Currently, we have no information to speculate
about the stereospecificity of the compounds.Some interesting
considerations can be done on the base of the
obtained results. Neurounina-1 and compounds 1–3, which present a methylene group as the spacer and a pyrrolidine
or piperidine ring as R, showed the same conformational preference,
characterized by the g–and anticonformation
of τ1 and τ2, respectively (Table ). The formation of
a six-membered intramolecular hydrogen bond between the protonated
nitrogen of R and the carbonyl oxygen of the benzodiazepinone ring
represented the driving force for the resulting conformational behavior
(Figure A). Accordingly,
the selectivity of compound 1 toward NCX1[27] compared to Neurounina-1, which
stimulates both NCX1 and NCX2,[25] is not
due to conformational effects but to the steric hindrance introduced
by the chlorine atom at the ortho position of the pendant phenyl ring,
which is not tolerated by the NCX2 binding site (Figure D).
Figure 8
(A) Neurounina-1 and compound 1 (NCX1
activators); (B) compound 14 (NCX1/3 activator); and
(C) compound 4 NCX1 (inhibitor); pharmacophore features:
aromatic ring (centroid and plane; green), hydrophobic-aliphatic group
(centroid; blue), hydrogen bond acceptor head/tail (balls and arrows;
green), distances (Å; black). (D–F): shape features. All
structures are superimposed by the carbon atoms of the benzodiazepinone
moiety and displayed as ball and sticks. Heteroatoms are colored by
atom type: carbon atoms are colored: Neurounina-1 and
compound 1, green; compound 4, orange; and
compound 14, magenta. Hydrogen atoms are omitted for
the clarity of presentation.
(A) Neurounina-1 and compound 1 (NCX1
activators); (B) compound 14 (NCX1/3 activator); and
(C) compound 4 NCX1 (inhibitor); pharmacophore features:
aromatic ring (centroid and plane; green), hydrophobic-aliphatic group
(centroid; blue), hydrogen bond acceptor head/tail (balls and arrows;
green), distances (Å; black). (D–F): shape features. All
structures are superimposed by the carbon atoms of the benzodiazepinone
moiety and displayed as ball and sticks. Heteroatoms are colored by
atom type: carbon atoms are colored: Neurounina-1 and
compound 1, green; compound 4, orange; and
compound 14, magenta. Hydrogen atoms are omitted for
the clarity of presentation.Importantly, the complete loss of activity of 3, presenting
a piperidine ring in place of the pyrrolidine substituent of Neurounina-1, revealed the very strict shape features required
by the NCX cleft accommodating the R group (Figure F). Finally, the inactivity of 2 indicated that the nitro substituent at the benzo-fused ring of Neurounina-1 is involved in key interactions with the protein
binding site, which cannot be any more established when it is replaced
by a chlorine atom. This suggested the involvement of the hydrogen
bond acceptor features of the nitro group at the benzo-fused ring
(Figure A–C).Interestingly, when the spacer of Neurounina-1 is
extended to an ethylene group, the resulting compound 4 was still able to bind to NCX1, but the effect changed from activation
to inhibition. In this case, the lowest energy conformational family
is characterized by the g+ conformation
of τ1 and the anticonformation of τ2, respectively (colored in orange in Figures A and 8C). Consequently,
as evidenced in Figure E, the pyrrolidine ring of the NCX inhibitor 4 is differently
oriented with respect to those of the activators Neurounina-1 and compound 1.This suggested that 4 binds to (stabilizes) a different
NCX1 conformation with respect to that bound by activators, in which
the protein binding partner of the pyrrolidine ring is the same, but
differently oriented. In support of this hypothesis, similarly to
what was observed for 3 with respect to Neurounina-1, regardless of preserving the same conformational preference (Table ), the enlargement
of the pyrrolidine ring of the NCX1 inhibitor 4 to a
piperidine ring (5) as well as to methyl-substituted
piperidine rings (6 and 7) resulted in inactive
compounds, thus confirming the very strict shape requirements for
the R group observed for activators.On the other hand, the
replacement of the pyrrolidine ring of 4 with an imidazole
ring (8) also led to a complete
loss of activity. This structural modification changed both the electronic
nature of R and the conformational preference of the resulting compound
(8; Table ; Figure B). The
same conformational preference is shared by 9–11 (colored in orange in Figure B), where the ethylene spacer is combined with other aromatic
R groups, which also resulted to be inactive. As evidenced in Figures and 8F, when compared to those of the active compounds, the R groups
of 8–11 occupy different regions of the space
with respect to the benzodiazepinone system.A third type of
spacer was tested by replacing a methylene unit
with a carbonyl group (12–19) and, by consequence,
changing the amine function of the R groups into an amide function,
not protonated at physiological pH.Due to the sp2 hybridization of the introduced carbonyl
group and the attraction between the carbonyl oxygen and the hydrogen
atoms of the pendant phenyl ring, the introduction of such a spacer
strongly affected the value of τ1 (about −120°;
family III, Table ) and, by consequence, the orientation of the R group (colored in
magenta in Figure ). In particular, the lowest energy conformational family of compounds 12–15 and 18–19 presented an anticonformation
of τ2 (family IIIc), while that of 16 and 17 presented a g+ conformation
of τ2 (family IIIa). Accordingly, the pyrrolidine
ring of 12 occupies a region other than those occupied
by the pyrrolidine rings of either NCX activators (Neurounina-1 and compound 1) or the NCX inhibitor 4 (Figures A and 8F). The same is valid for the bulkier R groups of
the other derivatives presenting the same spacer, with the only important
exception of compound 14.Interestingly, while
all the other analogues were inactive, 14 resulted in
an NCX activator with a peculiar activity profile.
Contrarily to the other active compounds, 14 showed selectivity
toward NCX1 and NCX3. Molecular volume comparisons showed that only
the methyl substituent on the piperidine ring of 14 is
able to partially cover the volume occupied by the pyrrolidine group
on Neurounina-1 and its active analogue 1 (Figure B). Therefore,
even though the region to be occupied by NCX1 and NCX3 activators
is not the same, it has to partially overlap to show any activity.Moreover, 14 was found to activate NCX1 with a potency
comparable to that of Neurounina-1 and compound 1; however, while these latter showed the same activity both
in the forward and reverse mode of action of the transporter, 14 resulted more effective in activating NCX1 working in the
reverse mode. On the contrary, in the case of NCX3, the compound is
more effective when tested on the forward mode. Thus, according to
its peculiar molecular shape (pharmacophore), 14 resulted
selective for a specific conformation of NCX1 and NCX3, namely, the
outward-facing (OF) bound to calcium and sodium ions, respectively
(see the next paragraph). Finally, because compound 14, contrarily to the other active derivatives, lacks the amine function,
the protonation of the R group is not necessary for both NCX1 and
NCX3 binding; however, this structural difference could contribute
to the selectivity profile showed by the compound.In summary,
the active compounds share the same pharmacophore moieties,
such as the benzodiazepinone scaffold, the nitro substituent on the
benzo-fused ring, and a nitrogen-containing aliphatic ring as R group
characterized by very strict steric requirements. The changing in
the nature of the spacer is responsible for the different pharmacophore
distances and molecular shapes, thus determining the different NCX
selectivities and intrinsic activity profiles showed by Neurounina-1, 1, 4, and 14.
Investigation
of the Putative Binding Site for Neurounina-1 and Its
Active Analogues
The atomic structure of the human
NCX transporters has not been experimentally determined, with the
exception of their intracellular calcium binding sites (CBD1 and CBD2)
on the large cytosolic regulatory loop (f-loop) between TM5 and TM6.[32,33] However, the structure of the homologous NCX transporter of ArchaebacteriumMethanococcus jannaschii (NCX_Mj) was crystallized in different conformational states of
the OF orientation (Na+-bound, Ca2+-bound, apo
form, open, semi-open, and occluded).[34,35] Indeed, as
demonstrated by kinetic analyses,[36−38] under steady-state conditions,
the membrane-bound NCX_Mj and its mammalian orthologues preferentially
adopt the OF orientation.Compared to human NCXs, NCX_Mj lacks
the f-loop but preserves the 10 membrane-spanning segments comprising
two homologous halves, which constitute the basic functional unit
for ion transport.[12,36,39,40] In particular, NCX_Mj preserves the highly
conserved α1- and α2-repeats, that is, internal repeat
sequences (presumably the product of intragenic duplication) located
between the putative transmembrane (TM) helices TM2 and TM3 and TM7
and TM8, respectively, and involved in ion translocation and apparent
ion affinity.[41−45] Three Na+ and one Ca2+ are transported in
separate steps, and the ion pocket encompasses the highly conserved
α1 and α2 repeats with inverted topology, with 12 ion-coordinating
residues and two helix-breaking signature sequences (on TM2C and TM7C)
each containing a highly conserved proline residue (NCX_Mj P53 and
P212, corresponding to P147 and P848 in hNCX1, respectively) actively
involved in ion transport activities.[41−45]Previous mutagenesis studies[25] allowed
the identification of two regions within the NCX1 α1- and α2-repeats
involved in Neurounina-1 binding (namely, NCX1_alpha1_Neu:
aa141–180 and NCX1_alpha2_Neu: aa842–879; hNCX1 numbering).
Sequence alignments show that these regions are conserved in all NCX
proteins and present full identity among human, dog, and rat (the
species used in the present study) while presenting some differences
among NCX1, NCX2, and NCX3 (Figure S4).
With the aim of identifying the NCX1 putative binding site for Neurounina-1 and its active analogues, we checked the similarity
of NCX1_alpha1_Neu and NCX1_alpha2_Neu with the binding motifs contained
in experimentally determined benzodiazepine binding sites by performing
a structural and bioinformatic analysis (see the Experimental Section for details). Results evidenced a significant
(>30%) similarity with the following: (1) the GSK525762A (BET-I)
binding
site (within 5 Å from the ligand) on the human BRD4 bromodomain
(PDB ID: 3P5O);[46] (2) the flurazepam binding site on
the pentameric ligand-gated ion channel from Erwinia
chrysanthemi (ELIC) (Figures and S5).
Figure 9
(A) Calculated Neurounina-1 lowest energy conformation
(carbons: pink) superimposed on the X-ray structure of I-BET (carbons:
dark gray) in complex with the BRD4 bromodomain (PDB ID: 3P5O). The I-BET binding
site is colored in red (BRD4_BDZ_BS_1) and cyan (BRD4_BDZ_BS_2). P85
is evidenced in CPK. The pyrrolidine substituent of Neurounina-1 is indicated by a black arrow. (B) Sequence alignments of the α1
and α2 repeat regions suggested to be involved in Neurounina-1 binding with the BRD4 alprazolam binding site. BRD4 residues establishing
interactions with the benzodiazepine ligand are evidenced with red
squares. (C) Calculated Neurounina-1 lowest energy conformation
(carbons: pink) superimposed on the X-ray structure of flurazepam
(carbons: green) in complex with ELIC (PDB ID: 2YOE). The binding site
is colored in red (ELIC_BDZ_BS). P82 is evidenced in CPK. The pyrrolidine
substituent of Neurounina-1 is indicated by a black arrow.
(D) Sequence alignments of the α1 and α2 repeat regions
suggested to be involved in Neurounina-1 binding with
the ELIC flurazepam binding site. ELIC residues establishing interactions
with the benzodiazepine ligand are evidenced with red squares. NCX1
P848, BRD4 P82, and ELIC P85 proline residues are evidenced and labeled.
(A) Calculated Neurounina-1 lowest energy conformation
(carbons: pink) superimposed on the X-ray structure of I-BET (carbons:
dark gray) in complex with the BRD4 bromodomain (PDB ID: 3P5O). The I-BET binding
site is colored in red (BRD4_BDZ_BS_1) and cyan (BRD4_BDZ_BS_2). P85
is evidenced in CPK. The pyrrolidine substituent of Neurounina-1 is indicated by a black arrow. (B) Sequence alignments of the α1
and α2 repeat regions suggested to be involved in Neurounina-1 binding with the BRD4 alprazolam binding site. BRD4 residues establishing
interactions with the benzodiazepine ligand are evidenced with red
squares. (C) Calculated Neurounina-1 lowest energy conformation
(carbons: pink) superimposed on the X-ray structure of flurazepam
(carbons: green) in complex with ELIC (PDB ID: 2YOE). The binding site
is colored in red (ELIC_BDZ_BS). P82 is evidenced in CPK. The pyrrolidine
substituent of Neurounina-1 is indicated by a black arrow.
(D) Sequence alignments of the α1 and α2 repeat regions
suggested to be involved in Neurounina-1 binding with
the ELIC flurazepam binding site. ELIC residues establishing interactions
with the benzodiazepine ligand are evidenced with red squares. NCX1
P848, BRD4 P82, and ELIC P85 proline residues are evidenced and labeled.In particular, the BRD4 I-BET binding site is composed
by two regions:
aa 81–97, herein named BDZ_BS_1 (colored in cyan in Figure ), and aa 135–149,
herein named BDZ_BS_2 (colored in red in Figure ). BRD4_BDZ_BS1, which contains the 81WPF83 I-BET interacting motif,[46] showed significant sequence similarity with NCX1_alpha2_Neu.
Importantly, the proline residue of the 81WPF83 motif (P82) resulted aligned with NCX1 P848 (human numbering), the
conserved proline residue within the signature sequence motif of the
α-repeats (Figure ).[34]On the other hand, BRD4_BDZ_BS2
resulted best aligned with NCX1_alpha2_Neu
(with which it shares a helix-loop-helix arrangement), showing the
conservation of some residues contacting the benzodiazepine ligand
but with an overall sequence similarity <30% (Figure ).
Figure 10
(A,B). Molecular interaction
model between Neurounina-1 and NCX_Mj resulting from
our bioinformatic and structural analysis.
The X-ray structure of the NCX_Mj transporter in the sodium-loaded
semi-open conformation (PDB ID: 5HWY) is colored in white with NCX1_alpha1_Neu
and NCX1_alpha2_Neu evidenced in cyan and yellow, respectively. The
protein structure is displayed as follows: helical structures as wide
cylinders, β-sheets as arrows, and coil and turn regions as
tubes. The sodium atoms are displayed in ball and stick and colored
in violet. The putative bioactive conformer of Neurounina-1 is displayed in ball and stick and colored by atoms (C = green,
O = red, and N = blue). Neurounina-1 solvent accessible
surface is showed and colored in white/transparent. Proline P212 is
evidenced in CPK and colored in orange. (C) Sequence alignments of
the α1 and α2 repeat regions of human NCX1 suggested to
be involved in Neurounina-1 binding with the corresponding
segments of NCX_Mj.[31] NCX_Mj P212 and NCX1
P848 proline residues are evidenced and labeled.
(A,B). Molecular interaction
model between Neurounina-1 and NCX_Mj resulting from
our bioinformatic and structural analysis.
The X-ray structure of the NCX_Mj transporter in the sodium-loaded
semi-open conformation (PDB ID: 5HWY) is colored in white with NCX1_alpha1_Neu
and NCX1_alpha2_Neu evidenced in cyan and yellow, respectively. The
protein structure is displayed as follows: helical structures as wide
cylinders, β-sheets as arrows, and coil and turn regions as
tubes. The sodium atoms are displayed in ball and stick and colored
in violet. The putative bioactive conformer of Neurounina-1 is displayed in ball and stick and colored by atoms (C = green,
O = red, and N = blue). Neurounina-1 solvent accessible
surface is showed and colored in white/transparent. Proline P212 is
evidenced in CPK and colored in orange. (C) Sequence alignments of
the α1 and α2 repeat regions of human NCX1 suggested to
be involved in Neurounina-1 binding with the corresponding
segments of NCX_Mj.[31] NCX_Mj P212 and NCX1
P848 proline residues are evidenced and labeled.The flurazepam binding site on ELIC (ELIC_BDZ_BS: aa72–107;
colored in red in Figure ) is formed by β-sheets and showed a significant sequence
similarity with both NCX1_alpha1_Neu and NCX1_alpha2_Neu. It also
included a proline residue (P85) in close contact with the benzodiazepine
ligand (Figure ).
Interestingly, also P85 of ELIC resulted aligned with NCX1 P848, the
key proline residue at the N-terminal end of the TM7B α-helix
of the NCX1 transporter (α2 repeat). Moreover, the superimposition
(by the common phenyl-benzodiazepine skeleton) of the energetically
most favored conformer of Neurounina-1 on the co-crystallized
ligands of the BRD4/BET-I and ELIC/flurazepam complexes, places the
key pyrrolidine R substituent in contact with P82 and P85, respectively
(Figure ).The
above results prompted us to speculate that the R groups of Neurounina-1 and its active analogues could establish a Pro–Pro-like
interaction with P848 on the α2-repeat of NCX transporters.This hypothesis was supported by the very strict conformational
and shape features required for the R substituent resulting from SAR
analysis. Because P848 (TM7B) in human NCX1 corresponds to P212 (TM7B)
in NCX_Mj (Figure C),[34] we challenged our hypothesis by
superimposing the heavy atoms of the P212 residue of NCX_MJ on the
heavy atoms of the P84 residue of the BET–I binding motif.
Strikingly, this placed the Neurounina-1 putative bioactive
conformer (previously superimposed on BET-I; Figure A) just behind the ion translocation pore
(Figure A,B).In particular, the pyrrolidine substituent resulted positioned
between the conserved helix-breaking regions of TM2B-C and TM7B-C,
while the 5-phenyl-1,4-benzodiazepinone scaffold was placed at the
interface between TM2/TM7 and TM1/TM6.Interestingly, other
positive allosteric modulators of transporters/channels,
such as Bay K 8644,[47] citalopram,[48] and ethanol,[49] characterized
by comparable or lower clog P/clog D7.4 values (Table S1), are
able to bind to similar sites close to the pore center.As evidenced
by our 3-D SAR analysis, depending on the different
linkers present in the structure, the R groups of Neurounina-1 and compounds 1, 4, and 14 assume different orientations with respect to the benzodiazepinone
skeleton. Accordingly, we have (i) NCX1 activators as Neurounina-1 (equally potent on the reverse and forward modes)[25] and compound 1 (more effective on the reverse
mode),[27] (ii) the NCX1 (reverse mode) inhibitor 4, and (iii) the NCX1 (reverse mode) and NCX3 (forward mode)
activator 14 (Figure ). According to the observed pharmacological profile,
the NCX1 inhibitor 4 should select/induce the OF calcium-loaded
occluded conformation of the transporter (i.e., working in the reverse
mode; PDB: 5HXR, Figure S6). Interestingly, the superimposition
of 4 on Neurounina-1 in the NCX_Mj (PDB: 5HXR) interaction model
placed the R group of 4 at the center of the ion channel
(Figure S6). On the other hand, in the
case of 14, the R group pointed more downward and resulted
closer to TM7C-B compared to Neurounina-1 (Figures S7 vs 10). NCX
activators should select/induce the open state of the transporter,
in particular, according to the observed pharmacological profile, 14 should preferentially bind/stabilize NCX1 in the OF calcium-loaded
open conformation (i.e., working in the reverse mode; Figure S7) and NCX3 in the OF sodium-loaded open
conformation (i.e., working in the forward mode).The transition
(open) state of NCX transporters has not been characterized;
however, it should be situated between the two OF and inward-facing
(IF) occluded states.[50] The symmetry of
NCX_Mj and its ion-binding sites, as well as the bidirectional ion
exchange, suggests that the IF model should maintain similar symmetry
and ion accessibility but with ion-permeation pathways directed toward
the intracellular side, analogous to an inverted structure of the
OF state (Figure S8).[34,51] It was proposed that in the transition (open) state, the ion bound
species stabilize the hydrophobic patch between TM2C (P53) and TM7C
[P212, corresponding to P848 in human NCX1 (hNCX1)], allowing the
relocation (sliding) of the gating bundle (TM1/TM6) toward the OF/IF
swapping.[52] Accordingly, we hypothesize
that our compounds, by binding between TM2B-C and TM7C-B (NCX_Mj P212/hNCX1
P848) and occupying the interface between TM2/TM7 and TM1/TM6, may
affect the interactions between the core domain and the gating bundle
and prevent (4) or favor (Neuronina-1, 1, and 14) the sliding motion of this latter.
In particular, our NCX activators could select/induce the open state
of the transporter: (i) by stabilizing a specific relative orientation
of the core domain with respect to the gating bundle corresponding
to the transition (open) state of the channel or (ii) by interacting
with TM2 and TM7 in place of TM1 and TM6, loosening the connection
between the core and the gating bundle, and lowering the energetic
barrier (ΔG) of the OF/IF transition. In support
of this hypothesis, close to the putative binding region of Neuronina-1 and its active analogues, we found a number of
protein–protein interaction motifs (LxxLL-like and heptad repeat),
whose interplay could drive the conformational transition. The identified
motifs are present on TM1, TM2, TM6, and TM7 of both NCX_Mj and mammalian
NCX transporters (Figure S9) and are conserved
among the NCX transporters used in the experimental studies (i.e., Canis lupus familiaris NCX1, Rattus NCX2, and Rattus NCX3) and their
human homologues.Although it is not appropriate to speculate
on the structural details
underlying the NCX subtype selectivity shown by our ligands based
on the experimental structure of NCX_Mj, nevertheless, some general
considerations can be made. With this aim, the differences in the
amino acid composition (excluding the f-loop) of the NCX isoforms
used in the experimental studies were mapped on the NCX_Mj structure.
Results showed that six residues are not conserved within the putative
ligand binding region (within 5 Å from any ligand atom), while
several residues are mutated at the interfaces between the TM domains
as well as in the connecting loops (Figures S10 and S11). Similar results were also obtained considering the
three human NCX isoforms (Figure S12).Subtype selectivity of ligands which bind among TM domains is strongly
affected by mutations of residues involved in interdomain interactions.[53] Accordingly, besides the residue differences
in the putative binding region, we hypothesize that the subtype selectivity
of Neurounina-1 and its active derivatives could depend
on a slightly different structural arrangement of the TM domains among
the three isoforms, which, in turn, could determine different ligand
binding pocket shapes, similarly to what was reported for G-protein-coupled
receptor ligands.[54]In summary, the
putative binding region on the NCX transporters
for Neurounina-1 and its active derivatives which resulted
from our bioinformatic and structural analysis could really account
for the observed pharmacological activities of the tested compounds
and matched the results of our 3-D SAR study.
Conclusions
In the present paper, we reported the synthesis and pharmacological
characterization of three functional modulators of the Na+/Ca2+ exchanger (compounds 1, 4, and 14), two of which are completely new (compounds 4 and 14). From the functional point of view,
compound 14 strongly activated NCX1 and NCX3 isoforms,
while compound 4 selectively inhibited NCX1. Interestingly
these compounds displayed a good propensity to differently modulate
Ca2+ and Na+ fluxes associated with the reverse
and forward modes of operation of each exchanger isoform. Accordingly,
compound 4 reduced the NCX1 reverse mode associated to
Ca2+ entrance/Na+ efflux with much more efficacy
than the forward mode associated to Ca2+ extrusion/Na+ entrance. On the other hand, besides its highest potency
in stimulating NCX1, compound 14 displayed a greater
efficacy for the NCX3 forward mode. However, only compound 1 showed a neuroprotective profile in primary cortical neurons exposed
to anoxic/hypoxic conditions. Therefore, we could speculate that a
good stimulatory agent of NCX with a neuroprotective profile might
have a greater ability to stimulate NCX1 or NCX3 reverse mode associated
to Ca2+ entrance/Na+ efflux and a less ability
to potentiate NCX3 forward mode associated to Ca2+ extrusion/Na+ entrance. In fact, our results suggest that the excessive
activation of NCX3 forward mode determining Ca2+ extrusion/Na+ entrance may negatively counterbalance the activation of
its reverse mode, favoring Ca2+ entrance/Na+ extrusion.The results of an in-depth conformational analysis
showed that
the different orientations of the R group of Neurounina-1, 1, 4, and 14 could account
for their different activities and selectivity profiles. Furthermore,
in agreement with our 3-D SAR analysis, the predicted binding site
of Neurounina-1 and its active analogues includes a conserved
proline residue responsible for key NCX conformational changes during
ion translocation. The ligand-based pharmacophore model and the molecular
interaction model between NCX_Mj and the analyzed active compounds
provide useful tools for the design of novel and specific NCX modulators
as potential neuroprotective agents. Future directions of the present
study will include the homology building of full length human NCX1-3
transporters and their use in dynamic molecular docking studies in
complex with Neuronina-1 and its derivatives.
Experimental Section
Materials and Methods
Nitrazepam and clonazepam were
purchased from F.I.S. (Fabbrica Italiana Sintetici); nordiazepam was
synthesized following an already published procedure;[55] all the reagents, solvents, or other chemicals were commercial
products purchased from Sigma-Aldrich. Solutions were concentrated
with a Buchi R-114 rotary evaporator at low pressure. All reactions
were followed by TLC carried out on Merk Silica Gel 60 F254 plates
with fluorescent indicator on the plates visualized with UV light
(254 nm). Preparative chromatographic purifications were performed
using the silica gel column (Kieselgel 60). Melting points, determined
using a Buchi melting point B-540 instrument, are uncorrected and
represent values obtained on re-crystallized or chromatographically
purified material. Mass spectra of final products were recorded on
a LTQ Orbitrap XL Fourier transform mass spectrometer equipped with
an ESI ION MAX (Thermo Fisher, San José, USA) source operating
in the positive mode. All synthesized compounds are >95% pure (elemental
analysis). Elemental analyses were carried out on a Carlo Erba model
1106; analyses indicated by the symbols of the elements were within
±0.4% of the theoretical values. NMR spectra were recorded on
a Bruker AVANCE Neo 700 (1H-700 MHz e 13C-175
MHz) instrument. All spectra were recorded in CDCl3. Chemical
shifts are reported in ppm. The following abbreviations are used to
describe peak patterns when appropriate: s (singlet), d (doublet),
dd (double doublet), t (triplet), and m (multiplet).
Commercially available
NaH (Merck, 60% suspension in mineral oil, 427 mg, 17.8 mmol) was
suspended on anhydrous DMF (40 mL) and stirred at 0 °C in a two-neck
flask. A solution of nitrazepam (FIS, 5 g, 17.8 mmol) in anhydrous
DMF (30 mL) was added dropwise over 15 min. After 30 min, a solution
of 1-bromo-2-chloroethane (1.78 mL, 21.3 mmol) in anhydrous DMF (5
mL) was added. The mixture was stirred at room temperature for 4 h.
Successively, 10 mL of H2O and 80 mL of ethyl acetate were
added. The mixture was extracted with brine (3 × 20 mL). The
organic phase was dried on anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by column chromatography
[dichloromethane/methanol 9.75:0.25 (v/v)]. The combined and evaporated
product fractions were crystallized from diethyl ether, yielding 2.95
g of the desired intermediate A as an orange solid. Yield:
59%. Anal. Calcd for C17H14ClN3O3: C, 59.40; H, 4.10; N, 12.22. Found: C, 59.51; H, 4.11; N,
12.26. 1H NMR (CDCl3): δ 8.42 (dd, 1H, J = 2.6, 9.1), 8.22 (d, 1H, J = 2.6), 7.62
(d, 2H, J = 7.4), 7.57 (d, 1H, J = 9.1), 7.53 (t, 1H, J = 7.4), 7.45 (t, 2H, J = 7.4), 4.92 (d, 1H, J = 10.7), 4.76
(m, 1H), 4.01 (m, 1H), 3.80 (d, 1H, J = 10.7), 3.77
(m, 1H), 3.69 (m, 1H). ESI-MS: 344.7[M + H]+; 366.4 [M
+ Na]+.
Commercially available NaH (Merck, 60% suspension in mineral oil,
427 mg, 17.8 mmol) was suspended on anhydrous DMF (40 mL) and stirred
at 0 °C in a two-neck flask. A solution of nitrazepam (FIS, 5
g, 17.8 mmol) in anhydrous DMF (30 mL) was added dropwise over 15
min. After 30 min, a solution of tert-butyl bromoacetate
(3.16 mL, 21.4 mmol) in anhydrous DMF (5 mL) was added dropwise. The
mixture was stirred at room temperature for 4 h. Successively, 10
mL of H2O and 80 mL of ethyl acetate were added. The mixture
was extracted with brine (3 × 20 mL). The organic phase was dried
on anhydrous Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography [n-hexane/ethyl acetate 5:5 (v/v)]. The combined and evaporated product
fractions were crystallized from diethyl ether, yielding 3.87 g of
the desired intermediate B as a yellow pale solid. Yield:
59%. Anal. Calcd for C21H21N3O5: C, 63.79; H, 5.35; N, 10.63. Found: C, 64.03; H, 5.35; N,
10.67. 1H NMR (CDCl3): δ 8.40 (dd, 1H, J = 2.6, 9.1), 8.23 (d, 1H, J = 2.6), 7.62
(d, 2H, J = 7.4), 7.53 (t, 1H, J = 7.4), 7.45 (overlapped, 2H), 7.44 (overlapped, 1H), 4.97 (d, 1H, J = 10.7), 4.65 (d, 1H, J = 17.2), 4.33
(d, 1H, J = 17.2), 3.88 (d, 1H, J = 10.7), 1.50 (s, 9H). ESI-MS: 396.3[M + H]+; 418 [M
+ Na]+.
Intermediate C (300 mg, 0.88 mmol)
was dissolved in anhydrous dichloromethane (10 mL) in a two-neck flask.
An excess of SOCl2 (642 μL; 8.80 mmol) was added.
The mixture was heated at reflux for 1 h. The solvent was evaporated
under reduced pressure. The light yellow residue was dissolved in
dichloromethane and re-evaporated three times. The residue was used
in the next step without further purification. The acyl chloride was
obtained as a light yellow powder and was dissolved in anhydrous tetrahydrofuran
(10 mL). Imidazole (120 mg, 1.66 mmol) was added, and the reaction
was stirred overnight at room temperature. The reaction resulted in
the precipitation of imidazolium chloride. The mixture was filtered
and the solvent was evaporated under reduced pressure, yielding the
crude product. Crystallization from diethyl ether and n-hexane yielded 149 mg of the desired product 16 as
an intense yellow solid. Yield: 48%. Anal. Calcd for C20H15N5O4: C, 61.69; H, 3.88; N, 17.99.
Found: C, 61.83; H, 3.89; N, 18.04. mp 120–121 °C. 1H NMR (CDCl3): δ 8.33 (dd, 1H, J = 2.6, 9.1), 8.20 (d, 1H, J = 2.6), 7.60 (d, 2H, J = 7.4), 7.53 (t, 1H, J = 7.4), 7.40 (t,
2H, J = 7.4), 7.31 (s, 1H), 7.02 (d, 1H J = 9.1), 6.72 (s, 1H), 6.63 (s, 1H), 4.92 (d, 1H, J = 10.7), 4.85 (d, 1H, J = 16.7), 4.38 (d, 1H, J = 16.7) 3.78 (d, 1H, J = 10.7). 13C NMR (CDCl3): δ 169.2, 168.8, 165.1, 148.3,
143.2, 137.8, 136.4, 131.0, 129.5, 129.4, 128.7, 126.1, 126.0, 123.0,
116.2, 56.6, 50.4. ESI-MS: 390.3 [M + H]+.
Baby hamster
kidney (BHK) cells stably expressing dog cardiac NCX1 and NCX3 were
grown as previously reported.[57] For video-imaging
and electrophysiological studies, cells were seeded on glass coverslips
coated with poly-l-lysine (Sigma, St. Louis, MO).
Rat
Primary Cortical Neurons
Rat cortical neurons were
prepared and grown as previously reported.[22,23] During the exposure to hypoxic conditions, primary cortical neurons
at 7/10 DIV were incubated with different concentrations of each previously
identified compound. Italian Ministry of Health and the local Animal
Care Committee of “Federico II” University of Naples
(Italy) approved all animal procedures adopted (D. Lgs. March 4th,
2014 from Italian Ministry of Health; DIR 210/63 UE; 12/2018-UT7).
High-Throughput Screening to Test the Biological Activity with
Fluorescent Probes
High-throughput assay was aimed to screen
libraries of compounds for their activity on the NCX reverse mode.
This analysis was performed by microplate screening applications in
semi-automated experiments measuring changes in the intracellular
Ca2+ level after the addition of Na+-free solution
to BHK-NCX1 and BHK-NCX3 cells loaded with Fluo-4AM or Fura 2AM. Before
testing the compounds, cells were separately loaded with Fluo-4 AM
or Fura 2AM (10 μM each for 30′ at 37 °C). Identification
of newly synthesized compounds enhancing NCX reverse mode or inhibiting
NCX activity was done by measuring their ability to increase, or decrease,
the Na+-free-dependent Ca2+ level above the
mean of basal value of almost ±2 S.D.
[Ca2+] Measurement on
Single Cell
[Ca2+] was measured by single-cell computer-assisted video-imaging using
Fura-2 acetoxymethyl ester (Fura-2AM). Neurons were loaded with Fura
2AM (10 μM for 30′ at 37 °C). Experiments were carried
out with a MicroMax 512BFT cooled CCD camera (Princeton Instruments,
Trenton, NJ, USA), LAMBDA 10-2 filter wheeler (Sutter Instruments,
Novato, CA), and Meta-Morph/MetaFluor Imaging System software (Universal
Imaging, West Chester, PA). Fura-2 fluorescence intensity was measured
every 3 s. From each coverslip, 40–60 individual cells were
selected and monitored simultaneously. Assuming that the KD for FURA-2
was 224 nM, the equation of Grynkiewicz was used to for calibration.[58]The NCX reverse mode was determined by
switching the normal Krebs medium to Na+-deficient NMDG+
medium (Na+-free), as previously reported.[57]
Patch-Clamp Electrophysiology
All
currents were recorded
by patch-clamp technique in the whole-cell configuration using a Digidata
1322A interface (Molecular Devices). Data were acquired and analyzed
using pClamp software (version 9.0, Molecular Devices). The currents
were recorded by fire-polished borosilicate electrodes with a final
resistance of 2.5–4 MΩ filled with a specific internal
solution.NCX currents filtered at 5 kHz were recorded from
BHK wild type (Wt), BHK-NCX1, and BHK-NCX3 stably transfected cells.[29] Briefly, NCX currents were recorded starting
from a holding potential of −60 mV up to a short-step depolarization
at +60 mV (60 ms).[15,29] Then, a descending voltage ramp
from +60 to −120 mV was applied. The current recorded in the
descending portion of the ramp (from +60 to −120 mV) was used
to plot the current–voltage (I–V) relation curve. The magnitudes of NCX currents were measured
at the end of +60 mV (reverse mode) and at the end of −120
mV (forward mode), respectively.The Ni2+-insensitive
components were subtracted from
total currents to isolate NCX currents. The external Ringer solution
contained (in mM) the following: 126 NaCl, 1.2 NaHPO4,
2.4 KCl, 2.4 CaCl2, 1.2 MgCl2, 10 glucose, and
18 NaHCO3 adjusted to pH 7.4 with NaOH. Moreover, 20 mM
tetraethylammonium (TEA), 10 nM tetrodotoxin (TTX), and 10 μM
nimodipine were added to the external solution to block delayed outward
rectifier KV, L-type CaV, and TTX-sensitive NaV channels. The dialyzing
pipette solution contained (mM) the following: 100 K-gluconate, 10
TEA, 20 NaCl, 1 Mg-ATP, 0.1 CaCl2, 2 MgCl2,
0.75 EGTA, and 10 HEPES, adjusted to pH 7.2 with CsOH. TEA and Cs
were included in the dialyzing solution to block delayed outward rectifier
K+ components. The values of NCX currents were normalized
for membrane capacitance.
Oxygen and Glucose Deprivation
Hypoxic
conditions were
induced by exposing rat primary cortical neurons to a oxygen- and
glucose-free medium in a humidified atmosphere containing 95% nitrogen
and 5% CO2, as previously reported.[22,23] Cell injury was assessed by MTT analysis. Briefly, neurons were
incubated with 1 mL of MTT solution (0.5 mg/mL in PBS). After 1 h
of incubation at 37 °C, neurons were dissolved in dimethyl sulfoxide
and the rate of MTT variation was measured spectrophotometrically
at the wavelength of 540 nm.[22,57]
Chemical
Hypoxia and RX
Chemical hypoxia was reproduced
by exposing primary cortical neurons for 45 min to a glucose-free
medium containing oligomycin (5 μg/mL) plus 2-DG (2 mM), followed
by RX (3 h). The hypoxic medium was composed of (in mM) 145 NaCl,
5.5 KCl, 1.2 MgCl2, 1.5 CaCl2, and 10 Hepes,
pH 7.4. Control neurons were exposed to the neuronal growth medium.
Cell injury was assessed by MTT analysis at the end of each experiment.
Drugs, Statistical Analysis, and Determination of EC50’s and IC50
Compounds were solubilized
in dimethyl sulfoxide at concentrations of 1 mM, and stock solutions
were kept at −20 °C. Appropriate drug dilutions were prepared
daily. Statistical analysis was performed with two-way ANOVA, followed
by the Newman–Keuls test. Statistical significance was accepted
at the 95% confidence level (p < 0.05). Values
are expressed as means ± SEM. To obtain EC50’s
of newly synthesized compounds and IC50 of compound 4,
all data were fitted to the following binding isotherm: y = max/(11X/IC50 or EC50), where X is the drug concentration
and n is the Hill coefficient.
Molecular
Modeling
Molecular modeling calculations
were performed on an E4 Server Twin 2 x Dual Xeon-5520, equipped with
two nodes. Each node: 2 x Intel Xeon QuadCore E5520-2.26GHz, 36 GB
RAM. The molecular modeling graphics were carried out on a personal
computer equipped with an Intel Core (TM) i7-4790 processor and SGI
Octane 2 workstations.The apparent pKa and log D values (pH 7.4 and 7.2) of all
compounds were calculated by using the ACD/pKa classic algorithm of ACD/Percepta software (ACD/Percepta,
Advanced Chemistry Development, Inc., Toronto, ON, Canada, 2017, http://www.acdlabs.com). Then,
the percentage of neutral/ionized forms was computed at pH 7.4 (blood
pH value) and pH 7.2 (cytoplasm pH value) using the Handerson–Hasselbalch
equation. The compounds were considered in their prevalent (>50%)
ionic form at the considered pHs in the subsequent calculations.BIOVIA Discovery Studio 2017 was used to perform the calculations
and to generate the graphical results (DassaultSystèmes Vélizy-Villacoublay
France).Neurounina-1 and compounds 1–19 were built using the Small Molecule tool, and atomic potentials
and charges were assigned using the CFF forcefield.[59]The conformational space of the generated structures
was sampled
using the stochastic conformational search algorithm BEST for the
random generation of a maximum of 1000 conformations (Search Small
Molecule Conformations; Discovery Studio 2017). An energy threshold
value of 106 kcal/mol was applied as selection criteria
in order to ensure a wide variance of the resulting structures. The
generalized Born implicit solvent model with a solvent dielectric
constant value of 80 was used to mimic an aqueous environment.[60] The generated conformations were then subjected
to energy minimization until the maximum RMS derivative was less than
0.01 kcal/Å using conjugate gradient as the minimization algorithm.[61] The resulting conformers were ranked by their
potential energy values [that is, energy difference from the global
minimum (ΔEGM)] and the low-energy
conformers (ΔEGM ≤ 5 kcal/mol)
were analyzed.First, the structures were grouped according
to the flip of the
benzodiazepinone ring system, that is, positive or negative value
of the torsional angle related to the rotation around the CH2–C=O bond and having at the 1.4 positions the nitrogen
atoms (namely, τflip = ±65°). This generated
two sets of specular conformers having the same energy values and
the opposite sign of their torsion angles (for chiral compounds, they
corresponded to the conformers of the two configurational enantiomers).Then, the two resulting sets were classified into families according
to the values of the first two dihedral angles of the spacer (namely,
τ1 and τ2). Family classification
corresponded to the following criteria: I (−30° < τ1 < −90°), II (30° < τ1 < 90°), III (−90° < τ1 <
−150°), IV (90° < τ1 < 150°),
a (30° < τ2 < 90°), b (−30°
< τ2 < −90°), c (τ2 ∼ ±180°), d (τ2 ∼ 0°),
e (−90° < τ2 < −150°),
and f (90° < τ2 < 150°).The
ranges of torsional angle values refer to the conformers with
τflip = −65°, those applied to the specular
conformers (τflip = 65°) present the same absolute
values with the opposite sign. Resulting conformational families of
each compound were ranked according to their ΔEGM values and analyzed by fitting the following: (i) the
centroid of the benzo-fused ring, (ii) the centroid of the pendant
phenyl ring, (iii) the carbonyl oxygen atom of the benzodiazepinone
ring, and (iv) the first carbon atom of the spacer group. Pharmacophore
points and shape features were generated by using the Pharmacophores
module of Discovery Studio 2017. Molecular shapes were combined by
using Boolean operators.
Bioinformatic and Structural Analysis
The sequences
of sodium/calcium exchanger 1 of C. lupus familiaris (entry P23685), sodium/calcium exchanger 1 of Homo
sapiens (entry P32418), sodium/calcium exchanger 2
of Rattus (entry P48768), sodium/calcium
exchanger 2 of H. sapiens (entry Q9UPR5),
sodium/calcium exchanger 3 of Rattus (entry P70549), and sodium/calcium exchanger 3 of H. sapiens (entry P57103) were downloaded from the
UniProtKB/Swiss-Prot Data Bank (http://www.uniprot.org).NCX sequence alignments were
performed using the PROMALS3D server (http://prodata.swmed.edu/promals3d/promals3d.php)[62] using the following parameters: identity
threshold: 0.6; weight for sequence-based constraints: 1; weight for
amino acid scores: 0.8; weight for predicted secondary structure scores:
0.2; PSI-BLAST iteration number: 3; PSI-BLAST e-value inclusion threshold:
0.001; identity cutoff below which distant homologues are removed:
0.25; maximum number of homologues kept for each blast run: 300; PSI-BLAST
e-value cutoff against structural database: 0.001; and identity cutoff
below which 3D structure templates are not used: 0.2.Secondary
structure predictions were performed using the PredictProtein
server (www.predictprotein.org).[63]The presence of linear functional
motifs, such as heptad repeats
and LxxLL-like protein recognition motifs were predicted using the
following consensus sequences: (i) heptad repeat: [VLIFYWM]xx[VLIFYWM]xxx[VLIFYWM]xx[VLIFYWM]xxx;
(ii) LxxLL-like motifs: [VLIFYWM]xx[VLIFYWM][VLIFYWM], [VLIFYWM][VLIFYWM]xx[VLIFYWM],
[VLIFYWM] xx[VLIFYWM] [VLIFYWM]xx[VLIFYWM] (Predict Sequence Properties
protocol, BIOVIA Discovery Studio software, Dassault Systèmes).Experimentally determined structures of the following: (i) human
bromodomain-containing protein 4 (BRD4) in complex with I-BET (PDB
ID: 3P5O)[64] and with alprazolam (PDB ID: 3U5J);[65] (ii) pentameric ligand-gated ion channel from E. chrysanthemi (ELIC) in complex with flurazepam
and bromoflurazepam (PDB IDs: 2YOE and 4A98);[66] and (iii)
GABAA receptor in complex with diazepam and GABA (PDB: 6HUP)[67] were downloaded from the Protein Data Bank (PDB, http://www.rcsb.org/pdb/) and
analyzed using the Macromolecules tool of Discovery Studio 2020 (DassaultSystèmes
BIOVIA, San Diego). The sequences of the NCX1 α1- and α2-repeats
reported to be involved in Neurounina-1 binding (i.e.,
aa141–180 and aa842–879, respectively[25]) were aligned with the sequences of the benzodiazepine
binding sites (i.e., residues with at least one atom within 5 Å
from any ligand atom) of the above-listed PDB complexes.Sequence
alignments were performed using the BIOVIA Discovery Studio
2020 software (Dassault Systèmes) using the following parameters:
Pairwise Alignment Algorithm: Slow; Scoring Matrix: BLOSUM 30; Gap
Open Penalty: 1; Gap Extension Penalty: 0.01; Gap Penalty 5.0; and
Window Size: 5. The lowest energy conformer of Neurounina-1 was superimposed on BET-I (3U5J) and flurazepam by fitting the heavy atoms of the
common benzodiazepine skeleton.The X-ray structures of the
sodium/calcium exchanger from M. jannaschii (PDB IDs: 3V5U, 5HYA, 5HXC, 5HXE, 5HXR, 5HXS, 5HXH, 5HWX, 5HWY, 5JDF, 5JDG, 5JDH, 5JDL, 5JDM, 5JDN, and 5JDQ)[34,35] were downloaded from
the PDB and analyzed using the Macromolecules tool of Discovery Studio
2020 (DassaultSystèmes BIOVIA, San Diego). In order to obtain
molecular models of interaction with M. jannaschii NCX, first, the heavy atoms of the proline residue P82 of BRD4 (3U5J) were superimposed
on the heavy atoms of the proline residue P212 of Mj_NCX X-ray structures
(PDB IDs: 5HWY, 5JDF, and 5HYA), corresponding
to P848 in human NCX, and then, the calculated lowest energy conformation
of Neurounina-1, 4, and 14 on
the X-ray structure of alprazolam by the common phenyl-benzodiazepine
skeleton.The amino acids not conserved at the level of 10 TM
helices (TM1–TM10)
among the three different isoforms of NCX (i.e., NCX1 Canis, NCX2
Rat, and NCX3 Rat) were displayed on the molecular interaction model
between Neurounina-1 and M. jannaschii NCX (PDB ID: 5HWY) by comparing the alignments of the NCX sequences (isoforms and
species) with that published by Liao et al. between M. jannaschii NCX and human NCX1.[31]The topology model of the IF sodium/calcium exchanger
from M. jannaschii (PDB ID: 5HWY) was generated by
superimposing the α
carbon atoms of the terminal residues of helices 2B, 2C, and 3A on
those of helices 7B, 7C, and 8A, as described by Liao et al.[34]
Authors: Debora A Nicoll; Michael R Sawaya; Seunghyug Kwon; Duilio Cascio; Kenneth D Philipson; Jeff Abramson Journal: J Biol Chem Date: 2006-06-14 Impact factor: 5.157
Authors: Valeria de Rosa; Agnese Secondo; Anna Pannaccione; Roselia Ciccone; Luigi Formisano; Natascia Guida; Roberta Crispino; Annalisa Fico; Roman Polishchuk; Antimo D'Aniello; Lucio Annunziato; Francesca Boscia Journal: EMBO Mol Med Date: 2019-01 Impact factor: 12.137
Figure 1
Scheme 1
Scheme 2
Scheme 3
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10