The inhibitory role of curcumin derivatives on AMPA receptor subunits and their effect on the gating biophysical properties

Article abstract of DOI:10.1016/j.ejps.2019.06.005

Curcumin is a natural polyphenol that has a broad spectrum of therapeutic characters, including neuroprotective actions against various neurological diseases. However, the molecular mechanism behind its neuroprotective properties remains obscure. The curr

Full text of DOI:10.1016/j.ejps.2019.06.005

Accepted Manuscript
The inhibitory role of curcumin derivatives on AMPA receptor
subunits and their effect on the gating biophysical properties
Mohammad Qneibi, Othman Hamed, Oswa Fares, Nidal Jaradat,
Abdel-Razzak Natsheh, Qais AbuHasan, Nour Emwas, Rana Al-
Kerm, Rola Al-Kerm
PII:
S0928-0987(19)30214-3
https://doi.org/10.1016/j.ejps.2019.06.005
PHASCI 4951
DOI:
Reference:
To appear in:
European Journal of Pharmaceutical Sciences
Received date:
Revised date:
Accepted date:
5 April 2019
17 May 2019
3 June 2019
Please cite this article as: M. Qneibi, O. Hamed, O. Fares, et al., The inhibitory role of
curcumin derivatives on AMPA receptor subunits and their effect on the gating biophysical
properties, European Journal of Pharmaceutical Sciences, https://doi.org/10.1016/
j.ejps.2019.06.005
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ACCEPTED MANUSCRIPT
The Inhibitory Role of Curcumin Derivatives on AMPA Receptor subunits and Their
Effect on the Gating Biophysical Properties.
a,
b
b
c
Mohammad Qneibi *, Othman Hamed , Oswa Fares , Nidal Jaradat , Abdel- Razzak
d
a
a
b
b
Natsheh , Qais AbuHasan , Nour Emwas , Rana Al-Kerm , Rola Al-Kerm
a
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, An-Najah
National University, Nablus, Palestine.
b
Department of Chemistry, Faculty of Science, An-Najah National University, Nablus, Palestine.
c
Department of Pharmacy, Faculty of Medicine and Health Sciences, An-Najah National
University, Nablus, Palestine.
d
Department of Computer Information Systems, Faculty of Engineering and Information
Technology, An-Najah National University, Nablus, Palestine.
*
Corresponding author. Tel. +00-972-545 975 16; fax. +00-970-9 2345 982; E-mail address:
mqneibi@najah.edu; Postal address: P.O.Box 7 Nablus-Palestine.
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ABSTRACT
Curcumin is a natural polyphenol that has a broad spectrum of therapeutic characters, including
neuroprotective actions against various neurological diseases. However, the molecular
mechanism behind its neuroprotective properties remains obscure. The current study investigated
the neuroprotective properties of 7 different curcumin derivatives on the gating biophysical
properties of AMPA receptors, specifically on the calcium-permeable homomeric GluA1 and
calcium impermeable heteromeric GluA1/A2 subunits. Due to the association between excessive
activation of AMPARs and neurotoxicity linked to numerous pathologies, we aim to target and
manipulate the kinetics of AMPARs through these derivatives. The current study used patch-
clamp electrophysiology to measure the whole-cell currents in the presence and absence of the
curcumin derivatives onto HEK293 cells expressing AMPA subunits. Our results showed that
some of the curcumin derivatives showed an inhibitory effect and altered the gating biophysical
properties, namely, deactivation and desensitization. In the presence of those derivatives, the
peak current measured was significantly reduced, and the desensitization and deactivation rates
decreased as well, achieving slower kinetics of the receptor and depressing its activity. These
results suggest that the two most promising derivatives have inhibitory actions and act as
allosteric modulators. Many neurological diseases like Epilepsy, ALS, and strokes are associated
with overactivation of AMPA receptors. We can potentially synthesize a more potent
neuroprotective drug to treat those neurological diseases, by understanding the most stable
chemical interaction between the derivative and the receptor underlying the reported neuronal
depressive properties.
Keywords: Neuroprotective; Curcumin Derivatives; AMPA Receptors; Activation;
Desensitization; Deactivation.
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1
. Introduction
Curcumin is a natural polyphenol that has various effects on the body including anti-
inflammatory, antioxidant, anti-cancer, and cardio-protective properties (Aggarwal et al., 2004;
Hatcher et al., 2008; Maheshwari et al., 2006; Shishodia et al., 2005; Singh et al., 1998).
Moreover, it has various effects on the central nervous including neuroprotective effects such as
anti-protein-aggregation also has antioxidant, and anti-inflammatory activities (Bertoncello et
al., 2018; Menon and Sudheer, 2007). Curcumin showed protective properties against glutamate
excitotoxicity, which has been linked to various neurological diseases (Chen et al., 2015;
Jayanarayanan et al., 2013; Matteucci et al., 2011) including epilepsy, Amyotrophic Lateral
Sclerosis (ALS), Alzheimer’s disease (AD), and strokes (Chang et al., 2012).
Glutamate, which is involved in neurotoxicity, mediates the majority of excitatory
neurotransmission in the central nervous system (CNS) (Traynelis et al., 2010) and targets two
families of receptors; the metabotropic (mGluRs) and ionotropic (iGluRs) Glutamate receptors
(Simeone et al., 2004). The agonists AMPA, NMDA and Kainate target the three types of
receptors composing iGluRs, which are α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
receptor (AMPAR), N-methyl-D-aspartate receptor (NMDAR) and Kainate receptor (Kew and
Kemp, 2005). Although AMPA, kainite, and NMDA receptors share a similar amino acid
sequence, they diverge in function (Herguedas et al., 2016). In addition, AMPAR mediates most
of the fast-excitatory neurotransmission (Collingridge et al., 2009; Esteban, 2003) and plays a
critical role in neuronal development and brain activities, such as learning and memory
formation (Sanderson et al., 2008). Moreover, the activation of AMPARs are on a microsecond
domain time scale, yet undergoes profound desensitization on the millisecond timescale. The
kinetics of AMPARs, not only shape synaptic plasticity, determining long-term potentiation or
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long-term depression, but also act as a neuroprotective mechanism against neurotoxicity (Kew
and Kemp, 2005). These key features of AMPARs make them the focal point of interest in
understanding various pathologies related to glutamate neurotoxicity as well as identifying
inhibiting compounds that can modulate the kinetics of the receptor.
The composition of AMPARs at the synapse play a major role in its function depending on its
pore-forming subunits (GluA1-4). Functional AMPA receptors are formed by the assembly of
tetramers of one or more subunits or isoforms (Hollmann and Heinemann, 1994; Seeburg, 1993).
The topology of any AMPA subunit can be divided into three domains; i. four hydrophobic
domains, M1-M4 of which one is a re-entry loop, M2, while the rest are transmembrane
domains, ii. a short intracellular cytoplasmic C-terminus and iii. An extracellular domain that
consists of the N-terminal domain and the ligand binding domain. The segments of the
extracellular domain between M3 and M4 are termed as S1 and S2 respectively have been shown
to determine the specificity of agonist binding to AMPARs. (Herguedas et al., 2016; Schwenk et
al., 2012; Sobolevsky et al., 2009; Sun et al., 2002).
Neurologic diseases have been treated using pharmacological drugs, however, despite modest
efficacy, these drugs have several side effects including extrapyramidal symptoms, akathisia,
tardive dyskinesia or the development of more serious conditions such as cardiac arrhythmias,
severe neuroleptic syndrome and stroke (Brodaty et al., 2003; Ma et al., 2014). While AMPARs
were studied, many ligands have been shown to possess selective inhibitory properties against
them (Libbey et al., 2016; Nikam and Kornberg, 2001; Qneibi et al., 2012), therefore, efforts
have been made to discover antagonists against AMPARs with better potency, specificity and
longer duration of action.
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Heterocyclic compounds are distinct regarding their biological activities, and they comprise a
common structural unit of most marketed medicines. The capability of the drug in forming
hydrogen bonds improves its solubility in hydrophilic solvents and the ability to interact with the
bioactive sites. Given these facts and as a part of our ongoing studies on developing a new
natural based drug, we decided to prepared curcumin derivatives with various heterocyclic
moieties and evaluated their inhibitory role on AMPA Receptor. The type and size of a
heterocyclic ring, in addition to the ring substituents, impact the biological activity of the
compound (Broughton and Watson, 2004) strongly, three different types of heterocyclic
moieties pyrazole, isoxazole, and diazepine was chosen for this purpose in addition to a Schiff
base. These curcumin derivatives are accessible to synthesis and in a quantitative yield. Besides,
benzodiazepines derivatives have been shown to exhibit desirable anticonvulsant and
neuroprotective properties in cellular and animal models (1). Several pyrazole derivatives
showed an excellent binding affinity for AMPA receptor (2). Also, compounds with isoxazole
moiety showed high potency and selectivity toward AMPA antagonists (3). Regarding the Schiff
base, the carbonyl groups in curcumin compounds are weak H-bonding acceptor, and one way of
enhancing its potency and binding affinity toward AMPAR is by adding functionality that acts
as an H-bonding donor and acceptor. The current study aims to investigate the effect of
curcumin derivatives on the calcium impermeable homomeric GluA1 and calcium permeable
heteromeric GluA1/A2 AMPA subunits in addition aims to assess the changes in the whole cell
currents and AMPARs biophysical properties.
2
. Results and discussion
2
.1. Effect of curcumin derivatives on the peak current of AMPAR subunits
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The effect of curcumin derivatives on the whole-cell current amplitude (A) was measured in the
absence and presence of curcumin derivatives (A ). To ensure an efficient, low-noise whole-cell
I
recordings, the amplitude of both GluA1 and GluA1/A2 receptors was measured using Integrated
Patch Amplifiers (IPA). For both AMPA receptors’ subunits, we chose 10 mM for the high
glutamate concentrations. This concentration corresponds to ∼95% of the open-channel form
(Qneibi et al., 2012). The Agonist was applied on AMPAR by using Fast Exchange solution i.e.
Piezo. Cell currents were measured in the presence and absence of curcumin derivatives at a
concentration of 20 uM, no change in the inhibition nor the biophysical properties above this
concentration was noted. Data were analyzed using Igor 7 software.
For GluA1 homomer, the peak current for glutamate in the absence of any curcumin derivative
was 774±69 pA. Curcumin derivative CR-MeNH significantly decreased the peak current to
1
51±13 pA and CR-NO decreased it to 146±11 pA. While CR-NN and CR-NNPh showed the
similar effect as CR-NN decreased peak current to 188±39 pA and CR-NNPh decreased it to
02±37 pA. However, curcumin, CR-PhCl, CR-PhF and CR-PhBr did not show significant
change. The peak currents for those derivatives were 528±45 pA, 759±38 pA, 766±26 pA, and
51±38 pA, respectively.
For GluA1/A2, the peak current for glutamate in the absence of any curcumin derivative was
10±53 pA. Curcumin derivative CR-MeNH significantly decreased the peak current to 54±8.0
3
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pA and CR-NO decreased it to 55±6.4 pA. While CR-NN and CR-NNPh showed the similar
effect as CR-NN decreased peak current from to 75±7.0 pA and CR-NNPh decreased it to
1
05±13 pA. However, curcumin, CR-PhCl, CR-PhF and CR-PhBr did not show significant
change. The peak currents for those derivatives were 220±31 pA, 298±27 pA, 304±41 pA, and
98±26 pA, respectively.
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A
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Fig. 2. Effect of curcumin and the derivatives on the amplitude of the whole-cell current with
and without application of the derivatives.
The graphs (right)illustrate the peak current for the whole cell recordings of GluA1 (A) and
GluA1/A2 (B) conducted at −60 mV, pH 7.4, and 22 °C, upon 500 ms application of 10 mM
glutamate with and without curcumin derivatives. While on the left are above G current traces
from HEK293 cells expressing homomeric GluA1 (A) and heteromeric GluA1/2 (B). For both A
and B, the most potent derivatives to reduce peak current are CR-NN, CR-NNPh, CR-MeNH and
CR-NO. Patches were repeated 5-7 times ± SEM.
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2
.2. Curcumin derivatives alter AMPAR desensitization rate
The average desensitization time of GluA1 was 3.1±0.1ms denoting an average rate (τ=1/ms) of
-1
0
.32 ms . The derivative CR-MeNH and CR-NO significantly increased the average
-1
desensitization time from 3.1±0.1 ms to 9.2±0.7 ms decreasing the rate from 0.32 ms to 0.11
-1
ms by ~2.9 folds, while CR-NO increased the desensitization time to 9.4±0.9ms, decreasing it
-1
rate 0.11 ms by ~2.9 folds. Both CR-NN and CR-NNPh showed similar effects. CR-NN
increased the average desensitization time from 3.1±0.1 ms to 8.4±0.7 ms decreasing the
-1
desensitization rate to 0.12 ms by ~2.6 folds and CR-NNPh increased the desensitization time
-1
to 7.1±0.5 ms decreasing its rate to 0.14 ms by ~2.3 folds. However, curcumin, CR-PhCl, CR-
PhF and CR-PhBr did not show significant change. The average desensitization time for those
derivatives was 3.9±0.9ms, 2.9±0.9ms, 2.9±0.8 ms and 3.2±0.7 ms, respectively.
For GluA1/A2, the average desensitization time was 5.4±0.6 ms with a rate of 0.19 ms-1.
Curcumin derivative CR-MeNH significantly increased the average desensitization time from
-1
-1
5
.4±0.6ms to 13.2±2.9ms decreasing the rate from 0.19 ms to 0.08 ms by ~2.4 folds while
CR-NO increased the average desensitization time to 14.4±3.4 ms, decreasing the desensitization
-1
rate to 0.07 ms by ~2.7 folds. Both CR-NN and CR-NNPh showed similar effects. CR-NN
increased the average desensitization time from 5.4±0.6ms to 10.2±1.4ms decreasing the rate
-1
-1
from 0.19 ms to 0.1 ms by ~1.9 folds and CR-NNPh increased the desensitization time to
-1
8
.7±1.2ms, decreasing the rate to 0.11 ms by ~1.7 folds. However, curcumin, CR-PhCl, CR-
PhF and CR-PhBr did not show significant change. The average desensitization time for those
derivatives was 5.4±0.6 ms, 5.2±0.9 ms, 5.2±0.4 ms and 5.6±0.3 ms, respectively.
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Fig. 3. Impact of curcumin and the derivatives on AMPAR desensitization.
Graphs summarize weighted time constants for desensitization (τ des) for the whole cell
W
recordings of GluA1 (A) and GluA1/A2 (B) conducted at −60 mV, pH 7.4, and 22 °C, upon 500
ms application of 10 mM glutamate with and without curcumin derivatives. For both A and B,
the most potent derivatives to reduce the desensitization rate are CR-NN, CR-NNPh, CR-MeNH
and CR-NO. Patches were repeated 5-7 times ± SEM.
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2
.3. Curcumin derivatives modify AMPAR deactivation rate
The effects of curcumin on AMPAR’s desensitization kinetics motivated the investigation of
Curcumin derivatives on AMPAR deactivation (Fig. 4). The average deactivation time for
-1
GluA1 was 2.4±0.1ms which denotes a rate of 0.42 ms . Curcumin derivative CR-MeNH
significantly increased the average deactivation time from 2.4±0.1ms to 7.3±0.6ms, decreasing
-1
-1
the deactivation rate from 0.42 ms to 0.14 ms by ~3 folds, while CR-NO increased it to
-1
7
.5±0.7ms, decreasing the rate to 0.13 ms by ~3.2 folds. CR-NN and CR-NNPh showed the
similar effect as CR-NN increased the average deactivation time from 2.4±0.1ms to 6.5±0.4ms
-1
-1
decreasing the deactivation rate from 0.42 ms to 0.15 ms by ~2.8 folds and CR-NNPh
-1
increased the deactivation time to 5.1±0.4ms, decreasing the rate to 0.2 ms by ~2 folds.
However, curcumin, CR-PhCl, CR-PhF and CR-PhBr did not show significant change. The
average deactivation time for those derivatives was 3.1±0.3ms, 2.5±0.6ms, 2.3±1.0ms and
2
.6±0.4 ms, respectively.
For GluA1/A2, the average deactivation time was 2.3±0.1ms with an average deactivation rate of
-
1
0
2
.43 ms . The derivative CR-MeNH significantly increased the average deactivation time from
-1
-1
.3±0.1 ms to 8.3±0.8 ms decreasing the rate from 0.43 ms to 0.12 ms by ~3.6 folds and CR-
-1
NO increased the deactivation time to 8.8±0.7 ms, decreasing the rate to 0.11 ms by ~3.9 folds.
While CR-NN and CR-NNPh showed similar effects as CR-NN increased the average
deactivation time from 2.3±0.1 ms to 7.8±0.6 ms decreasing the rates from 0.43 to 0.13 by ~3.3
folds and CR-NNPh decreased the deactivation time to 6.6±0.9 ms, decreasing its rate to 0.15 by
~2.9 folds. However, curcumin, CR-PhCl, CR-PhF and CR-PhBr did not show significant
change. The average deactivation time for those derivatives was 2.8±0.3 ms, 2.2±0.5 ms, 2.1±0.7
ms and 2.5±0.5 ms, respectively.
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Fig. 4. Results of Curcumin and the derivatives effect on AMPAR deactivation. The Graphs on
the right summarize weighted time constants for deactivation (τ deact) for the whole cell
W
recordings of GluA1 (A) and GluA1/A2 (B) conducted at −60 mV, pH 7.4, and 22 °C, upon 500
ms application of 10 mM glutamate with and without curcumin derivatives. While on the left are
above G current traces from HEK293 cells expressing homomeric GluA1 (A) and heteromeric
GluA1/2 (B). For both A and B, the most potent derivatives to reduce deactivation rates are CR-
NN, CR-NNPh, CR-MeNH and CR-NO. Patches were repeated 5-7 times ± SEM.
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The activation of AMPARs results in transforming a chemical signal into an electrical one, as it
takes place after the binding of glutamate to the receptor leading to the opening of a
transmembrane ion channel and allowing ion influx into the neuron. The work of Armstrong and
Mayer in 2004 provided structural data at atomic resolution for S1 and S2 domain, which
revealed their separation by the membrane regions M1 to M3. Moreover, it was demonstrated
that in an intact receptor the functional folding of AMPARs created two globular domains,
referred to as D1 and D2, extracellularly that play a major role in both activation and
desensitization. Hence, accumulating evidence shows the rotational changes between the dimers
upon agonist binding, which in return, influence the activation of the receptor. The structural
mechanism of channel opening and activation upon agonist binding to S1 and S2 has been
widely accepted to be associated with conformational changes in the extracellular domains. Yet,
the mechanism controlling the transduction of these conformation changes to channel gating
remain to be obscure. Although, it is believed to involve linker regions between TMD and LBD,
namely the S1-M1, S2-M3, and S2-M4 linkers (Balannik et al., 2005; Twomey and Sobolevsky,
2
018; Wang et al., 2014; Yelshanskaya et al., 2016).
Rearrangement of the extracellular dimer interface has also been identified in relation to the
desensitization of the receptor. Unlike deactivation, which is the natural decay in current after a
short simulation time following activation at which the ligand unbinds from the receptor,
desensitization is a long simulation time decay in current following long activation with the
ligand remaining to be bound to the receptor (Akhondzadeh and Abbasi, 2006; Vickers et al.,
2
001). As a consequence the flow of ion into the neuron through AMPARs is terminated via
deactivation or desensitization. A structural mechanism of desensitization was provided by Yu
Sun in 2002, which explained that the conformation stability of desensitization is higher than that
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of the activation state. It was found that upon agonist binding, the opening of the channel was
coupled with a separation of the linker regions, yet this required a stable dimer interface(Sun et
al., 2002; Twomey and Sobolevsky, 2018). Correctly, the channel opens as the gate recieves a
conformational strain, which has been caused by domain closure, due to agonist binding.
However, during desensitization, the dimer interface rearranges, thererfore no observable
conformational strains are being transmitted to the ion channel gate, as a result, the domain
closure is decoupled from the channel gate. Both desensitization and deactivation are potential
targets to manipulate the strength of AMPAR signal transmission, to treat various diseases that
are associated with excessive acitvation and intense signalling transduction from AMPARs. the
current study synthesize’s different curcumin derivatives as shown in Fig. 1 and investigate their
effects on the electrophysiology mediated by AMPA glutamate receptors. Their effects were
investigated using biochemical and electrophysiologic approaches by characterizing the effect on
AMPA receptors kinetics (i.e., peak current, desensitization, and deactivation), and detecting any
modulatory activity.
For both GluA1 homomer and GluA1/A2 heteromer, CR-MeNH and CR-NO showed the most
significant inhibitory effect by reducing the peak current by 5 folds. They also reduced both
deactivation and desensitization rates remarkably by at least 3 and 2 folds, respectively, denoting
the property to slow the kinetics of those receptors. Likewise, CR-NN and CR-NNPh had similar
impact on both receptors by decreasing the peak current and increasing the duration of the state
at which the receptor is desensitized or deactivated. However, Curcumin, CR-PhCl, CR-PhF and
CR-PhBr did not show significant changes in the peak current, deactivation or desensitization.
Our results regarding curcumin align with (Nelson et al., 2017) study that provides evidence to
classify curcumin as PAINS (pan-assay interference compounds) and an IMPS (invalid
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metabolic panaceas) candidate, as it had no effect on any of the tested biophysical properties nor
show any inhibition on either receptors.
The effect of CR-NN, CR-NNPh, CR-MeNH and CR-NO on the peak current of the receptor
reveal a decrease in activation, insinuating that the active sites of these compounds have an
affinity to an inhibitory binding site on the receptor. An increase in glutamate concentration had
no observable effect on derivative concentrations, meaning they act as noncompetitive inhibitors.
In accordance with various research on non-completive antagonist, such as, the derivatives of
2
,3-benzodiazepine, PMP, and CP, the interaction occurs in the linker regions, specifically S1-
M1 and S2-M4 linkers (Balannik et al., 2005) (Fig. S1).
As shown above, curcumin derivatives with five-membered ring heterocyclic moiety showed
excellent inhibition activities. The results could be explained based on structure activity-
relationship hypothesis.31 The size isoxazole and pyrazoles derivatives and the presence of the
heteroatoms (O, N) at positions 1 and 2 give the molecules the ability to accommodate well into
the binding pocket and interact with the receptor site as an H-bonding acceptor. Curcumin amino
compound CR-MeNH showed the highest inhibition activity. The size of the molecule and the
presence of the free amino groups give the molecule the ability to better fit in the binding pocket
and the strong capability to physically bond as H-bond acceptor and donor with the receptor site.
However, the benzodiazepines derivatives showed no activities. This could be attributed to the
hindrance of the molecule, due to the presence of the phenyl group, which is bonded from two
sites; 1 and 2. The hindrance is incapable of fitting into the binding site, thus affecting the
contact with the active functionality surrounding the active sites in the binding pocket. The
location of the heteroatoms at 1,4 positions, could affect the molecular contact between the
receptor sites of the pockets and the ring heteroatoms. Besides, the phenyl group acts as an
1
4

ACCEPTED MANUSCRIPT
electron withdrawing group, thus reducing the strength of H-bonding that might form between
the amino groups of the benzodiazepine and pocket active sites. Based on the obtained results
and the explanation we offered, we speculate that, compounds such as curcumin amine 2 (Fig. 1)
could show the highest binding potency and thus provide the highest inhibition activities.
Unfortunately, so far, we were not succeeding in preparing the curcumin amine compound.
The work of (Balannik et al., 2005) in mutagenesis research, an attempt to decode the structural
mechanism involved in noncompetitive inhibition, reveals the function of the inhibitors as
blocking the transmission of conformational changes in the LBD to D2, by acting as a wedge
(Twomey and Sobolevsky, 2018; Yelshanskaya et al., 2016). Moreover, as the inhibitor binds
entirely to the binding pocket of the receptor, conformational changes in the TMD is
accompanied, specifically at pre-M1/M1, M3, and M4, which have been further separated
(Yelshanskaya et al., 2016). Although various residues play a vital role in the binding pocket,
their role is dependent on the chemical structure of the inhibitor. Since CR-MeNH and CR-NO
had the most impact on both activation and desensitization, we assume the residues to be
involved are central for both activation and desensitization. Hence, the binding of the derivative
acts against the separation of linkers that are coupled with agonist binding for receptor opening,
but also stabilizing the desensitized and deactivated state of the receptors.
The results of the current study were independent of subunit makeup of the receptor as they
shared similar significant changes between the homomeric and heteromeric subunit. Previous
studies indicated that the residues on the AMPA receptor at the location of antagonist binding
seems to be shared throughout all of AMPA subunits; hence the binding affinity depends on the
chemical structure of the inhibitor (Balannik et al., 2005; Yelshanskaya et al., 2016). Depressing
the activity of those receptors has therapeutic effects on various disorders and CR-NN, CR-
1
5

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NNPh, CR-MeNH and CR-NO show such property that could be promising for future drug
synthesis. Moreover, the different results achieved using the curcumin derivatives shed light on
the possible effects of changing the chemical compositions and achieving a better understanding
of the receptors’ binding sites affinity to such changes.
3
. Conclusion
In conclusion, the current study demonstrated that the two-curcumin derivative CR-MeNH and
CR-NO had the most potent effects on AMPAR kinetics in comparison to the other tested
curcumin derivatives. The effects observed on AMPAR kinetics in our results revealed a
decrease in desensitization and deactivation rate, as well as a decrease in activation, suggesting
that the derivatives modify the AMPAR gating kinetics by the direct contact and association with
AMPA receptors. Furthermore, these derivatives act as noncompetitive inhibitors making AMPA
receptors less sensitive for its agonist glutamate. We propose three different mechanisms for the
observed effects of CR-MeNH and CR-NO on AMPAR kinetics; first, the derivatives might
affect AMPAR trafficking, reducing AMPAR density on the postsynaptic cleft. Second, they
might alter the chemical structure of the pore-forming groups of the AMPAR ion channel,
changing their electrochemical permeability to specific ions. Third, by acting as antagonistic
modulators, they bind to allosteric sites that, in return, affect AMPAR conformation, stabilizing
the desensitized and deactivation conformations and hindering activation. These results are
promising in comparison to alternative pharmacological treatment, as the derivatives of a natural
polyphenol will demonstrate lower side effects.
For future research, we aim to investigate the effects of these derivatives on calcium
impermeable GluA2 containing AMPA subunits and design a better understanding of
noncompetitive structural inhibition. This study specifically looks at curcumin derivatives
1
6

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influencing the biophysical gating properties of both homomeric and heteromeric AMPA
subunits, yet further research is needed to understand the specific interaction between the
receptor and the derivative and the mechanism of impact to potentially synthesize a non-
competitive inhibitory drug against AMPARs to treat the diseases that have been linked to
excessive AMPA activity.
4
. Experimental section
The curcumin derivatives chosen for this work and a future study (figure 1) are; 4,4'-((1E,1'E)-
isoxazole-3,5-diylbis(ethene-2,1-diyl))bis(2-methoxyphenol), 4,4'-((1E,1'E)-(1H-pyrazole-3,5-
diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol),
methylimino)hept-3-ene-1,7-diyl)bis(2-methoxyphenol),
4,4'-((3Z,5E)-3-(methylamino)-5-
4,4'-((1E,1'E)-(7-chloro-3H-
(
benzo[b][1,4]diazepine-2,4-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol),
fluoro-3H-benzo[b][1,4]diazepine-2,4-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol),
(1E,1'E)-(7-bromo-3H-benzo[b][1,4]diazepine-2,4-diyl)bis(ethene-2,1-diyl))bis(2-
4,4'-((1E,1'E)-(7-
4,4'-
(
methoxyphenol) and 1-phenyl-3,5-bis-2-(4-hydroxy-3-methoxystyryl)-1H-pyrazole, for the sake
of simplicity, the derivatives were abbreviated as; (CR-NO), (CR-NN), (CR-MeNH), (CR-PhCl),
(
CR-PhF), (CR-PhBr), and (CR-NNPh), respectively. The curcumin derivatives with five
membered heterocyclic rings were prepared according to a published procedure (Hamed et al.,
013).
2
1
7

ACCEPTED MANUSCRIPT
Fig. 1. The chemical structures of the prepared curcumin derivatives.
All chemicals used in this study were purchased from Aldrich Chemical Company and used as
1
13
they were received. All prepared compounds were characterized by MS/MS, H NMR, C NMR,
IR spectroscopy. Mass spectroscopic analysis was performed on solutions of compounds in a
Nano scale concentration using a multi dilution procedure. The MS/MS was performed on LCQ
Fleet ion trap mass spectrometer (Thermo Fisher Scientific, CA, USA) operated in a positive
electrospray mode. The electrospray voltage was 5 kV. The capillary temperature was 290°C
and the sheath gas flow was 30 units. An isolation width of 2 Da was used with a 20 msec
activation time for MS experiments. All scan events were acquired with a 250 ms maximum
ionization time. Nuclear Magnetic Resonance spectra were recorded on Varian Gemini 2000, 300
MHz instrument MHz instruments. Infrared spectra were recorded on a Shimadzu 820 PC FT-IR
spectrometer. Solvent used in the NMR was DMSO-d
6
, 1H NMR experiments were reported in δ
13
units, parts per million (ppm) downfield from tetramethyl silane (TMS). All C NMR spectra
1
8

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were reported in ppm relative to DMSO-d
6
(39.52 ppm). TLC analysis was performed on silica
gel plates pre-coated with Merck Kieselgel 60 F254 and visualization was done using UV lamp.
Samples purifications were carried out by either crystallization or by flash chromatography with
silica gel (100-200) mesh.
4
.1. General experimental procedure for the preparation curcumin with 5-membered ring
heterocyclic moiety
To a round bottom flask equipped with magnetic stirring bar and a condenser, curcumin (3.0
mmole, 1.1 g) was added followed with 20.0 mL acetic acid. The desired reagent of hydrazine
(3.0 mmole) or hydroxyl amine was added to the solution of curcumin. The reaction mixture was
refluxed for 6 h. The reaction mixture was concentrated in-vacuo, and re-dissolved in ethyl
acetate (50 mL). The ethyl acetate layer was washed with saturated solution of NaHCO3,
saturated solution of NaCl and water. Then it was dried over Na2SO4 and concentrated in-vacuo.
The produced solid was collected by suction filtration.
4
.2. 1-phenyl-3,5-bis-2-(4-hydroxy-3-methoxystyryl)-1H-pyrazole (CR-NNPh)
-1
Yield 71.6% (0.96 g), mp 127-129°C, IR: Vmax cm 3550 (-C-OH), 3345 (–C–NH), 3100, 1625
1
(
-C=N), 1610, 1585 (C=C, Ph) and 1080 (C-O ether) of (–O-CH
δ ppm: 3.93 (s, 6H, OCH
and, C6-H), 7.13 (d, 2H, J = 14.9 Hz, C1-H and C7-H), 7.15–7.33 (m, 8H, Ph-H), 7.45 (m, 2H,
3
). H-NMR (300 MHz, CDCl
3
)
3
), 6.07 (s, 2H, OH), 6.82 (s, 1H, C4-H), 7.05 (d, 2H, J = 14.9 Hz, C2-H
1
3
Ph-H), 7.72 (m, 1H, Ph-H). C NMR (300 MHz, CDCl3) δ ppm: (300 MHz, CDCl3): 56.1, 56.1,
1
1
01.2, 110.0, 111.0, 112.7, 116.1, 116.2, 117.5, 120.7, 125.2, 128.2, 128.3, 128.8, 129.0, 129.1,
29.1, 131.2, 133.3, 139.7, 142.8, 147.3, 147.8, 148.1, 148.3, 151.5, 161.2. Anal. Calcd for
C
27
H
24
N
2
4
O : C 73.62, H 5.49, N 6.36. Found: C 73.43, H 5.42, N 6.48.
1
9

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4
.3.
4,4'-((1E,1'E)-isoxazole-3,5-diylbis(ethene-2,1-diyl))bis(2-methoxyphenol) (CR-NO)
-1
Yield 72.0 (0.8 g), mp 116-119°C, IR: Vmax cm 3560 (-C-OH), 3035, 1640 (-C=N), 1605 (C=C,
1
1
7
586 (-C=C, Ph), 1330 (C-O of the five-member ring) cm-1. 1H-NMR (400 MHz, DMSO-d ):
6
H-NMR: δ 3.85 (s, 6H, 2OCH3), 6.25 (s, 2H, OH), 6.71 (s, 1H, C4-H), 6.84-7.01 (m, 3H),
.04-7.15 (m, 4H), 7.26 (m, 3H). 13C-NMR (300 MHz, CDCl ) δ ppm: 56.14, 56.18, 98.33,
3
1
1
10.56, 110.82, 113.12, 113.43, 115.98, 116.11, 116.24, 121.8, 122.16, 127.49, 127.83, 129.22,
35.27, 135.98, 148.22, 148.42, 162.70, 168.84. Anal. Calcd for C21H19NO5: C 69.03, H 5.24,
N 3.38. Found: C 68.89, H 5.21, N 3.41.
4
.4. 4,4'-((1E,1'E)-(1H-pyrazole-3,5-diyl)bis(ethene-2,1-diyl))bis(2-methoxyphenol) (CR-NN)
Yield 72.0 (0.8 g), mp 116-119°C, IR: 3545 (-C-OH), 3035, 1640 (C=N), 1608, 1560 (-C=C,
-1 1
Ph), 1331 (C-O of the five-member ring) cm . H-NMR (400 MHz, DMSO-d
6
): 1H-NMR: δ
3
7
.83 (s, 6H, 2OCH
3
), 6.25 (s, 2H, OH), 6.75 (s, 1H, C4-H), 6.81-7.07 (m, 3H), 7.07-7.16(m, 4H),
1
3
.31 (m, 3H). C-NMR (300 MHz, CDCl ) δ ppm: 56.1, 56.1, 98.4, 110.7, 110.8, 113.1, 113.5,
3
1
1
3
15.98, 116.1, 116.2, 121.8, 122.2, 127.6, 127.8, 129.2, 135.3, 135.8, 148.2, 148.4, 162.7,
68.85. Anal. Calculated for C21H19NO5: C 69.03, H 5.24, N 3.38. Found: C 68.89, H 5.21, N
.41.
4
.5. 4,4'-((3Z,5E)-3-(methylamino)-5-(methylimino)hept-3-ene-1,7-diyl)bis(2-
methoxyphenol) (CR-MeNH)
Curcumin (1.0 g, 2.7 mmol) was dissolved in 50 mL of ethanol. The mixture was stirred until a
clear solution was obtained. Methylamine solution 40% (0.465 g, 6 mmol) was added to the
solution and the reaction mixture was refluxed for 2 h. The solvent was evaporated under
vacuum, the residue was washed with 5% Na
2
CO
3
, water and dried. The residue was
recrystallized from ethanol/water solution (1:2 by volume). Yield 64% (0.68 g), mp 171-175 ºC.
2
0

ACCEPTED MANUSCRIPT
-1
IR: vmax cm 3602 (-C-OH), 3050 (=C-H), 2943 (C-H), 1630 (C=N), 1610 (-C=C), 1580 (C=C,
Ph), 1514, 1276, 1213, 1031. 1H-NMR (400 MHz, DMSO-d6): 1H-NMR (Fig. 2.13): δ 2.86 (s,
6
7
9
1
H, 2 NCH
3
), 3.83 (s, 6H, 2OCH
3
), 6.27 (s, 2H, OH), 6.69 (s, 1H, C4-H), 6.79-7.05 (m, 3H),
) δ ppm: 31.8, 39.6, 56.24, 56.17,
.06-7.19 (m, 4H), 7.28 (m, 3H). 13C-NMR (300 MHz, CDCl
3
8.41, 110.67, 110.93, 113.23, 113.31, 116.02, 116.32, 116.25, 121.91, 122.17, 127.52, 127.93,
29.19, 135.15, 136.06, 148.31, 148.43, 162.75, 168.8.
4
.6. DNA preparation
High-copy plasmid DNA (up to 20 μg) was prepared using QIAGEN Plasmid Mini Kit. A
selective plate was streaked followed by the selection of a single colony. LB medium containing
appropriate selective antibiotic was used for the starter culture inoculation. Incubation of the
culture was for approximately 8 hours at 37°C, later diluted in 3 ml selective LB medium, then
incubated at 37°C for 12-16 hours. The tube was centrifuged after which the formed pellet was
resuspended to harvest the bacterial cells. 0.3 ml of buffer P2 was added to the tube; then it was
inverted 4-6 times to homogenously mixing the contents. The tube was centrifuged and the
supernatant containing the plasmid DNA was obtained.
A QIAGEN-tip 20 was equilibrated using 1 ml buffer QBT; the column was left to empty by
gravity flow. The supernatant was added to the QIAGEN-tip 20, under gravity flow, it entered
the resin. Then the QIAGEN-tip 20 was washed with buffer QC. The DNA was eluted with 0.8
ml buffer QF. For precipitation, isopropanol was added, mixed and centrifuged, then
immediately and carefully, the supernatant was decanted. The resultant DNA pellet was washed
with Ethanol, centrifuged again and the supernatant was decanted. Finally, the pellet was air-
dried, and the DNA was re-dissolved in a suitable volume of buffer. To calculate DNA
concentration both Bot spectrophotometry at 260 nm and quantitative analysis on an agarose gel
2
1

ACCEPTED MANUSCRIPT
were used. The reliability of spectrophotometric DNA quantification was determined by A260
readings between the values of 0.1 and 1.0.
4
.7. HEK293 Cell Culture and Transfection
Human Embryonic Kidney cells 293 (HEK293) were grown in Dulbecco Modified Eagle
Medium (DMEM) (Sigma, USA) containing 10% FBS (fetal bovine serum), 0.1 mg/ml
streptomycin, and 1 mM sodium pyruvate (Biological Industries; Beit-Haemek, Israel). HEK293
cells were incubated at 37° C and 5% CO2 was supplemented to the medium (Li et al., 2003). It
was subcultured twice a week until cells reached pass #20. The transfection reagent used was
either jetPRIME (Polyplus: New York, NY) or Lipofectamine 2000 (Invitrogen; San Diego, CA)
(Huang et al., 2005). Cells were kept for 24 hours after transfection then replated on coverslips
coated with Laminin (1 mg/mL; Sigma, Germany) to use for electrophysiology recordings or
stereomicroscope imaging.
4
.8. HEK293 Cell Patch-Clamp Recordings
Using IPA (Integrated Patch Amplifier) (Sutter Instruments, Novato, CA) on the whole cell
configuration of the patch-clamp technique, HEK293 Cells were recorded 36-48 hours after
transfection, at a temperature of 22°C, the membrane potential of -60 mV. SutterPatch Software
v. 1.1.1 (Sutter Instruments) to digitize membrane currents for a short period. Sampling
frequency was set to 10 kHz, and the low-pass filter was set to 2 kHz. Borosilicate glass was
used to fabricate the Patch electrodes with a resistance of 2-4 MΩ. The extracellular solution
contained (values are in mM): 150 NaCl, 2.8 KCl, 0.5 MgCl2, 2 CaCl2, 10 HEPES adjusted to
pH 7.4 with NaOH. The pipette solution contains (values are in mM): 110 CsF, 30 CsCl, 4 NaCl,
0
.5 CaCl2, 10 Trypsin EDTA solution B (0.25%), EDTA (0.05%), 10 HEPES, adjusted to pH
2
2

ACCEPTED MANUSCRIPT
7
.2 with CsOH. Using double barrel glass (theta tube) glutamate and solutions of choice were
rapidly administered, the theta tube was mounted on a high-speed piezo solution switcher
Automate Scientific, Berkeley, CA). After expelling the patch from the electrode to estimate the
(
speed of solution exchange, the open tip potentials were recorded during the application of
solutions of different ionic strengths. The 10%–90% solution exchange was typically at 500 ms.
Data acquired were analyzed using Igor Pro7 (Wave Metrics, inc) (Li et al., 2003; Qneibi et al.,
2
019). AMPAR deactivation and desensitization were measured by applying glutamate (10 mM)
for 1 ms and 500 ms, respectively. AMPAR-current deactivation and desensitization were fitted
with two exponentials, and the weighted tau (τ w) was calculated as τ w = (τf x af) + (τs x as),
where af and as are the relative amplitudes of the fast (τf) and slow (τs) exponential component.
4
.9. Statistical Analysis
Significance compared with AMPAR expressed alone or with AMPAR+ Curcumin derivatives;
p-value (one-way ANOVA): * < 0.05, ** < 0.01, *** < 0.001, ns – not significant
Acknowledgment
The work provided in this manuscript was supported by a grant from the Palestinian Ministry of
Education and Higher Education (Grant number: ANNU-MoHE-1819-Sc017), thus the authors
would like to acknowledge them in addition to An-Najah National University.
2
3

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ACCEPTED MANUSCRIPT
Graphical abstract
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ACCEPTED MANUSCRIPT
Highlights
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Over-activation of AMPA receptors is associated with various neurological diseases such
ALS, AD, epilepsy and strokes.
The effect of newly synthesized curcumin derivatives on the homomeric GluA1 and
heteromeric GluA1/A2 AMPA subunits were evaluated, compassing effects on the whole
cell current as well as the unique biophysical properties of AMPA.
CR-MeNH and CR-NO increased desensitization and deactivation rates up to 3 folds, and
resulted in highest level of inhibition.
CR-NN, CR-NNPh decreased peak current by 4 folds, and prolonged both desensitization
and deactivation states of both receptors.
Curcumin, CR-PhCl, CR-PhF and CR-PhBr showed no significant impact on any of the
tested biophysical properties nor possess any inhibitory actions.
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