Curcumin is an inhibitor of calcium/calmodulin dependent protein kinase II

Article abstract of DOI:10.1016/j.bmc.2012.08.029

Calcium/calmodulin dependent protein kinase II (CaMKII) is involved in the mechanisms underlying higher order brain functions such as learning and memory. CaMKII participates in pathological glutamate signaling also, since it is activated by calcium influx through the N-methyl-d-aspartate type glutamate receptor (NMDAR). In our attempt to identify phytomodulators of CaMKII, we observed that curcumin, a constituent of turmeric and its analogs inhibit the Ca²⁺-dependent and independent kinase activities of CaMKII. We further report that a heterocyclic analog of curcumin I, (3,5-bis[β-(4- hydroxy-3-methoxyphenyl)ethenyl]pyrazole), named as pyrazole-curcumin, is a more potent inhibitor of CaMKII than curcumin. Microwave assisted, rapid synthesis of curcumin I and its heterocyclic analogues is also reported.

Full text of DOI:10.1016/j.bmc.2012.08.029

Bioorganic & Medicinal Chemistry 20 (2012) 6040–6047
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Curcumin is an inhibitor of calcium/calmodulin dependent protein kinase II
M. Mayadevi a, D. R. Sherin b, V. S. Keerthi b, K. N. Rajasekharan b, R. V. Omkumar a,
⇑
a Molecular Neurobiology Division, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala, India
b Department of Chemistry, University of Kerala, Kariavattom Campus, Thiruvananthapuram, Kerala, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Calcium/calmodulin dependent protein kinase II (CaMKII) is involved in the mechanisms underlying
higher order brain functions such as learning and memory. CaMKII participates in pathological glutamate
signaling also, since it is activated by calcium influx through the N-methyl-D-aspartate type glutamate
receptor (NMDAR). In our attempt to identify phytomodulators of CaMKII, we observed that curcumin,
a constituent of turmeric and its analogs inhibit the Ca2+-dependent and independent kinase activities
of CaMKII. We further report that a heterocyclic analog of curcumin I, (3,5-bis[b-(4-hydroxy-3-methoxy-
phenyl)ethenyl]pyrazole), named as pyrazole-curcumin, is a more potent inhibitor of CaMKII than curcu-
min. Microwave assisted, rapid synthesis of curcumin I and its heterocyclic analogues is also reported.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 20 April 2012
Revised 20 August 2012
Accepted 22 August 2012
Available online 30 August 2012
Keywords:
CaMKII
Kinase
Inhibitor
Curcumin
Pyrazole
Neuroprotection
1. Introduction
the enzyme into an active state.7,8 Once relieved of the inhibition,
CaMKII phosphorylates substrates which include receptors, ion
Calcium/calmodulin dependent protein kinase II (CaMKII) is a
major mediator of Ca2+ signaling, which is found in brain, heart
and other tissues. CaMKII is a multifunctional serine/threonine ki-
nase with a broad range of substrates. CaMKII comprises of a fam-
channels, cytoskeletal and scaffolding proteins and it could also
undergo autophosphorylation. The autophosphorylation at Thr286
converts the enzyme into an autonomously active state, the func-
tional consequence of which allows it to function as a memory
molecule.9 CaMKII translocates to postsynaptic density, where it
regulates many signaling pathways.10
CaMKII, being a downstream target of glutamate induced cal-
cium signaling, may also play a role in pathological glutamate sig-
naling. Conflicting data regarding the role of CaMKII in
promoting11 or inhibiting neuronal cell death12 have been re-
ported. Currently known CaMKII inhibitors are mainly used as
tools for studying the enzymatic function of the kinase. Among
these, the KN series, which includes KN62 and KN93, do not inhibit
the autonomous activity.13,14 The peptides derived from the
autoinhibitory region of CaMKII, such as AIP (autoinhibitory pep-
tide) or AC3, also serve as CaMKII inhibitors. CaMKIIN has been re-
cently identified as a CaMKII specific, natural inhibitor15,16 and
tatCN21 inhibitor is a peptide derived from the natural inhibitor.17
ily of isoforms,
distinct genes. The
whereas
and d forms are found in most tissues including brain.1
a, b,
c
and d, derived from four closely related but
a
and b forms are restricted to nervous tissue,
c
CaMKII is highly expressed in the forebrain and it forms two per-
cent of the total protein in the hippocampus.2 Many of the neuro-
nal functions such as neurotransmitter synthesis, regulation of ion
channels and many signaling pathways are influenced by CaMKII.
CaMKII plays a crucial role in the mechanisms that underlie long
term potentiation (LTP) which serves as a cellular model for learn-
ing and memory.3,4 CaMKII exists as a dodecameric holoenzyme,
whose activation is regulated by the amplitude and frequency of
Ca2+ spikes.5 Each isoform of CaMKII has an N-terminal catalytic
domain, followed by a regulatory domain and a C-terminal associ-
ation domain. The kinase domains form dimers in which the cata-
lytic domains are held together by antiparallel coiled-coil formed
by the regulatory domains retaining the enzyme in an inactive con-
CaMKIIN
roles of which are not very clear. During contextual memory for-
mation, the level of CaMKIIN mRNA expression increases in
a and CaMKIINb are endogenous CaMKII inhibitors, the
formation.6 The activation of the N-methyl-
D
-aspartate type gluta-
a
mate receptor (NMDAR) promotes an influx of Ca2+ activating
CaMKII. The binding of Ca2+/calmodulin disrupts the interaction
of the regulatory domain with kinase domain, thereby converting
amygdala and hippocampus.18 CaMKIINb protein level has also
been reported to be increased in amygdala and hippocampus dur-
ing contextual memory formation and its expression is transient.19
These findings indicate that CaMKIIN type inhibitors could func-
tion as plasticity related molecules.20 Recent findings of Vest
et al. implicated the inhibition of Ca2+-independent autonomous
⇑
Corresponding author. Tel.: +91 471 2529483; fax: +91 471 2346333.
E-mail address: omkumar@rgcb.res.in (R. V. Omkumar).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.029

M. Mayadevi et al. / Bioorg. Med. Chem. 20 (2012) 6040–6047
6041
CaMKII activity that is generated by Thr286 autophosphorylation as
the drug target for post-insult neuroprotection.21
Curcumin is a phytochemical derived from the rhizome of Cur-
cuma longa commonly known as turmeric. The bioactivity of curcu-
min has been under intense study in the past decade and several
reviews in this topic have appeared.22,23 Natural curcumin is a mix-
ture of three closely related linear diarylheptanoids, curcumin I–III
(1–3) (Fig. 1), present in the ratio 85:11:4 approximately. All the
three exist in the enol form exclusively. Extensive computational
studies24 have confirmed the predominance of the enol tautomer
and have also shown that in curcumin, the middle seven-carbon
chain is quite hydrophobic whereas the enolic and terminal pheno-
lic groups provide polarity to the molecule. The bioactivity profile
of curcumin includes function as an antioxidant,25 reduction of
cognitive deficits,26 lowering of amyloid accumulation,27 reduction
of inflammation,28 inhibition of angiogenesis,29 prevention of
tumorigenesis,30 control of diabetes,31 amelioration of neurological
diseases,32,33 and inhibition of protein tyrosine kinase and protein
kinase C activities.34 Neuroprotective action of curcumin has been
reported in transgenic animal model for Alzheimer’s disease.35
Curcumin has been reported to prevent scopolamine–induced
memory dysfunction by inducing an improvement in the mem-
ory-dependent learning in rats.36 In addition to the reports on
the neuroprotective activity of curcuminoids, a recent report37
has identified the ability of curcumin to reduce corticosterone-in-
duced elevated phosphorylation of CaMKII in primary hippocampal
neurons. More recently, the memory stimulation effect of a phen-
ylpyrazole-curcumin derivative called CNB-001 (4) (Fig. 2) has
been reported38 and this activity has been suggested to be medi-
ated through the activation of CaMKII. The above two reports indi-
cate that, whereas curcumin decreases the corticosterone-induced
elevated phosphorylation level of CaMKII, its closely related deriv-
ative CNB-001 is an activator of CaMKII. We have now investigated,
under defined set of conditions, the effect of natural curcumin I-III
(1–3) and synthetic curcumin I (1), along with two pyrazole deriv-
atives (4, 5) and an isoxazole derivative (6) of curcumin I, on the
kinase activity of CaMKII.
Figure 2. Chemical structure of phenylpyrazole-curcumin (4), pyrazole-curcumin
(5) and isoxazole-curcumin (6).
spectra were recorded on Bruker Avance DPX-300 and Avance-III
400 and FAB MS spectra were recorded on JEOL JMS 600H spec-
trometers. Element analysis was done on Elementar Vario EL III
elemental analyzer. Natural curcumin, containing the three curc-
uminoids, was a gift from Synthite Industries, India.
2.1.1. Separation of curcumin I-III (1-3)
The natural curcumin supplied as a fine powder was separated
into its three components by dry column flash chromatography39
on silica gel-G (TLC grade, Merck, India) using dichloromethane -
chloroform (1:1) for curcumin I, dichloromethane - chloroform
(1:2) for curcumin II and chloroform for curcumin III as eluents
(Scheme 1). The collected fractions were evaporated under reduced
pressure and the product so obtained was crystallized from chloro-
form - ethyl acetate (4:1).
2.1.2. Microwave assisted rapid synthesis of curcumin I (1)
Vanillin (0.304 g, 2 mmol), boric acid (0.2 g) and acetylacetone
(0.1 mL, 1 mmol) in DMF (1.0 mL) were mixed in a conical flask
and were subjected to 300 W microwave irradiation in a Samsung
800 W multimode domestic microwave oven for 2 min in such a
way that after each irradiation for 15 s, 15 s cooling time was given
to prevent boil offs. During all microwave irradiation, a beaker con-
taining water (50 mL) was used to absorb excess microwave en-
ergy. After microwave irradiation for 2 min, tetrahydroquinoline
(0.02 mL) and glacial acetic acid (0.06 mL) were added to the above
mixture and it was subjected to 300 W microwave irradiation as
above. After 20 min, which involved 10 min of total heating, the
reaction mixture thus obtained was worked up by adding to acetic
acid (20%, 10 mL) under rapid stirring and the mixture was further
stirred for 1 h. The precipitate so obtained was filtered and air
dried. It was purified by dry column flash chromatography using
TLC grade silica gel-G eluted with dichloromethane - chloroform
(1:1) (Scheme 1) to obtain curcumin I (1); yield 72%; mp 182-
184 °C. Lit. mp 182 °C.39 Anal. Calcd for C21H20O6: C, 68.28; H,
5.47. Found: C, 68.32; H, 5.31; UV kmax (methanol): 268 and
430 nm; IR (KBr): 3350, 2900, 2950, 1620, 1580, 1505, 1460,
2. Materials and methods
2.1. Chemistry
TLC and dry column flash chromatography were performed
using TLC grade silica gel-G, particle size 10–40 microns, 300 mesh,
from Merck, India. Melting points were determined by open capil-
lary method. FTIR spectra were recorded on a Jasco FTIR, NMR
1420, 1280, 1260, 1200, 1160, 1120, 1030, 964, 940, 910 cmÀ1
;
1H NMR (300 MHz, CDCl3): d 3.91 (s, 6H, OMe), 5.82 (s, 1H, 4H),
6.56 (d, J = 16 Hz, 2H, 2,6H), 6.9–7.2 (m, 6H), 7.58 (d, J = 16 Hz,
2H, 1,7H); FAB MS spectrum, m/z: 369.78 [M+H]+. Calcd. For
[C21H20O6+H]: m/z 369.14.
2.1.3. Synthesis of pyrazole-curcumins (3,5-bis[b-(4-hydroxy-3-
methoxyphenyl)ethenyl]pyrazoles)
Curcumin I, separated from the natural mix, was mixed with
phenylhydrazine (1 mmol each) in gl. acetic acid (2 mL) and the
Figure 1. Chemical structure of curcumin I (1) curcumin II (2) and curcumin III (3).

6042
M. Mayadevi et al. / Bioorg. Med. Chem. 20 (2012) 6040–6047
2.1.4. Synthesis of isoxazole-curcumin (3,5-bis[b-(4-hydroxy-3-
methoxyphenyl)ethenyl]isoxazole) (6)
Curcumin I, separated from the natural mix, was mixed with
hydroxylamine hydrochloride (1 mmol each) in gl. acetic acid
(2 mL) and the mixture was subjected to microwave irradiation
(300 W, 2 min) allowing 15 s heating-cooling cycles to prevent
boil-offs. Subsequently, the mixture was diluted with water
(10 mL), the precipitate was filtered and the crude product so ob-
tained was purified by dry column flash chromatography, using
TLC grade silica gel-G and chloroform - ethyl acetate (1:1) as eluent
(Scheme 1), to obtain isoxazole-curcumin (3,5-bis[b-(4-hydroxy-3-
methoxyphenyl)ethenyl]isoxazole) (6) as a reddish brown amor-
phous solid in 83% yield; mp 158–159 °C, Lit. mp 162 °C;40
172 °C.43 Anal. Calcd for C21H19NO5: C, 69.03; H, 5.24; N, 3.83.
Found: C, 68.83; H, 5.04; N, 3.66; 1H NMR (300 MHz, CDC13): d
3.95 (s, 6H); 6.48 (d, J = 15.6 Hz, 1H); 6.51 (s, lH), 6.78–7.11 (m,
8H), 7.59 (d, J = 15.9 Hz,1H). FAB MS (NBA matrix) m/z: 366.53
[M+H]+. Calcd for [C21H19NO5+H]: m/z 366.14.
2.2. Biological experiments and assays
Insect cell culture medium (IPL-41) was from Sigma Chemicals,
USA. Bac-to-bac baculovirus expression kit was from GIBCO-BRL/
Invitrogen, USA. [c-
32P] ATP was from Bhabha Atomic Research
Centre, India. pGEX-2T vector was from Amersham (GE).
2.2.1. Expression of GST-fusion protein of NR2A substrate
sequence in Escherichia coli
Using the construct in pGEX-2T vector, the fusion protein, GST-
NR2A (NR2A amino acid residues 1265-1301), was expressed in
Escherichia coli (BL21 (DE3) pLys strain) and was purified as de-
scribed previously.44 Glutathione was removed from the purified
GST-NR2A by PD-10 column. The purified fusion protein was used
for the phosphorylation assay. The fusion protein was run on SDS-
PAGE45 and the amount of protein was determined as described
previously.44
Scheme 1. (A) Synthesis of curcumin I (1). Reagents and conditions: (i) 1,2,3,4-
Tetrahydroquinoline and gl. HOAc/DMF, microwave 300 W, 10 min, 20% aq HOAc
(B) Separation of curcumin I (1) from natural curcumin mix and its conversion to
heterocyclic analogs (4–6). Reagents and conditions: (i) Dry column flash chroma-
tography on TLC grade silica gel-G, eluted with dichloromethane-chloroform (1:1)
(ii) PhNHNH2; H2NNH2.H2O or H2NOH.HCl, gl. HOAc, microwave, 300 W, 2 min,
water.
2.2.2. Expression and purification of
baculovirus and Sf21 or High Five cell system
-CaMKII was expressed using baculovirus expression system,
in monolayer cultures of Sf21 or High Five cells. The -CaMKII pro-
tein was purified by ion-exchange chromatography followed by
affinity chromatography as described previously.44,46 The purified
enzyme preparation showed a single major band of the expected
size on 10% SDS PAGE (Fig. 3A) and Western blot (Fig. 3B).47 The fi-
nal purified sample was concentrated using Amicon ultracentrifu-
gal devices with 100 kDa molecular weight cut-off by centrifuging
at 5000 Âg to get the required concentration.
a-CaMKII using
mixture was subjected to microwave irradiation (300 W, 2 min)
allowing 15 s heating-cooling cycles to prevent boil-offs. Subse-
quently, the mixture was diluted with water (10 mL), the precipi-
tate was filtered and the crude product so obtained was purified
by dry column flash chromatography, using TLC grade silica
gel-G and chloroform - ethyl acetate (2:1) as eluent (Scheme 1),
to obtain phenylpyrazole-curcumin (4) (3,5-bis[b-(4-hydroxy-3-
methoxyphenyl)ethenyl]-1-phenylpyrazole; CNB-001) as a reddish
brown amorphous solid in 87% yield; mp 88–89 °C. Lit. mp 89 °C.40
Anal. Calcd for C27H24N2O4: C, 73.62; H, 5.49; N, 6.36. Found: C,
73.48; H, 5.61; N, 6.28; IR (KBr): 3510, 3030, 1595, 1508, 1273,
a
a
2.2.3. Preparation of postsynaptic density (PSD) fraction from
rat brain
1029, 960, 761 and 696 cmÀ1 1H NMR (300 MHz, CDCl3): d 3.89
.
(s, 3H), 3.92 (s, 3H), 5.68 (broad, 2H), 6.71 (d, J = 15.9 Hz, 2H),
6.82–7.14 (m, 9H), 7.33–7.52 (m, 5H). FAB MS (NBA matrix) m/z:
441.58 [M+H]+. Calcd for [C27H24N2O4+H]: m/z 441.18.
The PSD fraction was prepared from five male Albino Wistar
male rats aged 40–45 days weighing an average of about 100 g.
The animals were maintained at the animal house of Rajiv Gandhi
Centre for Biotechnology and experiments conducted were in
accordance with guidelines of CPCSEA (Committee for the Purpose
of Control and Supervision of Experiments on Animals), India and
Institutional Animal Ethics Committee. Rats were sacrificed by cer-
vical dislocation. Brains were gathered immediately and were
homogenized in solution A (0.32 M sucrose, 1 mM sodium bicar-
Using hydrazine hydrate in the above reaction instead of phen-
ylhydrazine, pyrazole-curcumin (5) (3,5-bis[b-(4-hydroxy-3-
methoxyphenyl)ethenyl]pyrazole) was obtained as a pale yellow
amorphous solid in 84% yield, after purification by dry column
flash chromatography using TLC grade silica gel-G and chloroform
- ethyl acetate (2:1) as eluent (Scheme 1); mp 208-10 °C. Lit. mp
211-4 °C40,41; 215 °C.42 Anal. Calcd for C21H20N2O4: C, 69.22; H,
5.53; N, 7.69. Found: C, 69.39; H, 5.38, N, 7.74; IR (KBr): 1595,
bonate, 1 mM MgCl2, 0.5 mM CaCl2, 0.1 mM PMSF, 1 lg/mL leupep-
tin) in a Teflon glass homogenizer by 12 up and down strokes. 4 mL
of solution A was used per gram weight of the brain tissue. The
homogenate was diluted to 10% (weight/volume) in solution A
and was centrifuged at 1400 Âg for 10 min at 4 °C. The supernatant
was collected and stored. The pellets were resuspended in solution
1512, 1274, 1031 and 817 cmÀ1 1H NMR (300 MHz, CDCl3): d
.
3.93 (s, 6H), 6.57 (s, 1H), 6.89 (d, J = 16 Hz, 2H), 6.80–7.20 (m,
8H). FAB MS (NBA matrix) m/z: 365.71 [M+H]+. Calcd for
[C21H20N2O4+H]: m/z 365.15.

M. Mayadevi et al. / Bioorg. Med. Chem. 20 (2012) 6040–6047
6043
was terminated by the addition of 5
l
L of 5Â SDS sample buffer.
The samples were subjected to 10% SDS–PAGE. The dried gel was
exposed to phosphor screen. Single band corresponding to the
phosphorylated fusion protein could be visualized on scanning
the phosphor screen using the Bio-Rad Personal Molecular Imager
FX and the band intensities were quantitated using Quantity One
software (Bio Rad). Activity was calculated from the band intensity
values as described earlier.44 The data from at least three indepen-
dent experiments were plotted using Origin software to calculate
IC50 values.
2.2.6. Autophosphorylation of CaMKII
Thr286-autophosphorylation of CaMKII was carried out in a
reaction mix containing 50 mM Tris pH 8.0, 10 mM MgCl2,
1.3 mM CaCl2, 4000 Units/mL of CaM, 0.2 mg/mL BSA and 50
32P] ATP (1000–2000 cpm/pmol), with or without different con-
centrations of curcumin or its analogues in a total volume of 20 L.
Autophosphorylation assay was performed by pre-incubating the
purified WT- -CaMKII enzyme containing assay mixture without
ATP for 30 s at 30 °C followed by a 30 s phosphorylation reaction,
initiated by the addition of [
32P] ATP. The reaction was stopped
lM
[c-
l
a
c
-
by the addition of 5Â sample buffer and the samples were run
Figure 3. A. 10% SDSPAGE of purified
Coomassie Brilliant Blue and B. Western blot of purified
anti- CaMKII monoclonal antibody. MW represents molecular weight marker.
a
-CaMKII (4
l
g) after staining with
on SDS–PAGE and were processed as described in the previous sec-
a
-CaMKII (2 g) using
l
a
tion. The 50 kDa band corresponding to a-CaMKII was quantitated.
Autophosphorylation at Thr286 was confirmed by the generation of
Ca2+-independent activity.
A (10% weight/volume) by homogenization by three strokes and
were centrifuged at 710 Âg for 10 min. The two supernatants were
pooled and were centrifuged at 13,800 Âg for 10 min. The pellet
obtained was suspended in solution B (0.32 M sucrose, 1 mM so-
dium bicarbonate) in a ratio of 2.4 mL per gram weight of the brain
tissue. Sucrose gradient was prepared in a 40 ml tube by layering
10 mL each of 1.2 M sucrose, 1.0 M sucrose, and 0.85 M sucrose
containing 1 mM sodium bicarbonate. The gradient was consti-
tuted with highest concentration at the bottom and the lowest at
the top. The pellet suspended in solution B was layered on top of
the sucrose gradient and was centrifuged at 82,500 Âg for 2 h in
a swinging bucket rotor. After centrifugation the band between
1.2 M and 1.0 M concentrations was collected and was diluted with
solution B (6 mL per gram weight of the tissue). An equal volume of
1% Triton X-100 in 0.32 M sucrose, 12 mM Tris–HCl (pH 8.1) was
added and the mixture was stirred in the cold for 15 min. The mix-
ture was centrifuged at 36,800 Âg for 45 min. The pellet was sus-
pended in 3 mL of 40 mM Tris pH 8.0, and was used as the PSD
fraction.
2.2.7. Ca2+-independent activity
Autophosphorylation was carried out as described in the previ-
ous section using nonradioactive ATP. Reaction was stopped by
adding EGTA to a final concentration of 3 mM. The Ca2+-indepen-
dent activity of the autophosphorylated enzyme for substrate
phosphorylation was carried out as described previously, but keep-
ing EGTA concentration to 5 mM in excess of Ca2+
.
3. Results and discussion
3.1. Chemical synthesis
Several synthetic approaches to curcumins, their analogs and
derivatives, especially for investigating the biological activities,
have appeared.48 These methods generally involve prolonged heat-
ing for several hours. We have now shown that curcumin I (1)
could be synthesized (Scheme 1A) from vanillin, acetylacetone
and boric acid in a double Knoevenagel condensation under micro-
wave conditions in a shorter reaction duration.
We have further shown that the microwave assisted synthetic
approach also provided a rapid method for the synthesis of three
analogs of curcumin I: phenylpyrazole-curcumin (4) (CNB-001;
3,5-bis[b-(4-hydroxy-3-methoxyphenyl)ethenyl]-1-phenylpyra-
zole), pyrazole-curcumin (5) (3,5-bis[b-(4-hydroxy-3-methoxy-
phenyl)ethenyl]pyrazole) and isoxazole-curcumin (6) (3,5-bis[b-
(4-hydroxy-3-methoxyphenyl)ethenyl]isoxazole), starting from
curcumin I separated from natural curcumin mix (Scheme 1B), in
good yield in few minutes, instead of heating for 6–24 h as re-
ported in literature.40,42,43
2.2.4. Preparation of cytosolic fraction from rat brain
Albino Wistar male rat was sacrificed by cervical dislocation.
Forebrain was collected immediately and was homogenized in
20 mM Tris, pH 7.0, 0.1 mM DTT, 0.1 mM PMSF and 1X protease
inhibitor cocktail. The homogenate was clarified by centrifugation
at 100,000 Âg for 30 min. The supernatant was used as the cyto-
solic fraction.
2.2.5. Fusion protein phosphorylation
The purified GST-NR2A (NR2A amino acid residues 1265–1301)
was used as the substrate for CaMKII in an in vitro assay. The reac-
tion mix containing a total volume of 20
pH 8.0, 10 mM MgCl2, 12 M CaM or 2500 Units/mL of CaM,
0.2 mg/mL BSA, 1.3 mM CaCl2, 10 mM DTT and varying concentra-
tions of curcumin or its analogues was incubated with 0.02 g of
WT- -CaMKII for 1 min at 30 °C. Curcumin was dissolved in DMSO
lL including 50 mM Tris
3.2. Biological assay
l
3.2.1. Curcumin inhibits CaMKII activity
l
In our attempt to identify modulators of CaMKII from natural
products, we observed natural curcumin to be an inhibitor of the
kinase activity of CaMKII. We then separated the three components
of natural curcumin mix (Fig. 1). Activity of CaMKII was measured
using a protein substrate, GST-NR2A, which harbors the sequence
a
before addition to the reaction mix. Controls received the same
amount of DMSO. The reaction was initiated by the addition of
GST-NR2A and 50
tion mix was incubated for 1 min at 30 °C. The phosphorylation
lM [c-
32P] ATP (200–1500 cpm/pmol). The reac-

6044
M. Mayadevi et al. / Bioorg. Med. Chem. 20 (2012) 6040–6047
around the CaMKII phosphorylation site, Ser1291, in the sequence of
the NR2A subunit of NMDAR.49 The Ca2+-dependent activity of
purified CaMKII (Fig. 3) for the phosphorylation of a protein sub-
strate GST-NR2A, is inhibited by the components of natural curcu-
min, curcumin I (1) (Fig. 4), II (2) and III (3) (Table 1). The inhibitory
potency of the three components did not differ markedly, curcu-
min I being the most potent inhibitor among the three. Hence, de-
tailed investigations were carried out using curcumin I. To rule out
the possibility that curcumin I, II and III chromatographically sep-
arated from the natural curcuminoid mixture, may contain minor
amounts of any coeluted compounds which could be inhibiting
CaMKII, the inhibitory activity of synthetically prepared curcumin
I was checked. Curcumin I made synthetically (Scheme 1A) also
inhibited Ca2+-dependent activity of CaMKII (Table 1). Synthetic
curcumin I however showed only a slightly higher inhibitory po-
tency than that of curcumin I isolated from the natural mix. The
inhibition of CaMKII by curcumin I was also found to be partially
reversible (data not shown).
Table 1
Inhibition of Ca2+-dependent activity of purified CaMKIIa
Compound
IC50 lM
Curcumin Ib
Curcumin II
Curcumin III
Synthetic Curcumin I
21 3.5
24 7.0
37 19.6
13 4.5
a
The IC50 value for each experiment was analyzed separately and the mean and
standard deviation from a minimum of three experiments were calculated and are
shown in the table.
b
Indicates that the same data is shown in Table 2 also.
Table 2
Inhibition of activity of purified CaMKII by natural curcumin Ia
Type of CaMKII activity studied
IC50 lM
Ca2+-dependent activity of CaMKIIb
21 3.5
45 6.7
34 4.5
33 9.0
37 6.3
Ca2+-dependent activity of autophosphorylated CaMKII
Ca2+-independent activity of autophosphorylated CaMKII
CaMKII autophosphorylation
Ca2+-independent activity of T286D-
a-CaMKII
3.2.2. Effect of curcumin I (1) on autophosphorylation
Some of the inhibitors currently available inhibit only Ca2+
-
a
The IC50 value for each experiment was analyzed separately and the mean and
stimulated activity of CaMKII, but do not inhibit autonomous
activity of CaMKII.21 Curcumin is now seen to be effective as an
inhibitor of autophosphorylation of CaMKII as well. In spite of
the fact that autophosphorylation of CaMKII is essential for the
enzyme to function as a memory molecule, autophosphorylation
also occurs during restrained stress50 and after traumatic brain
injury51 in hippocampal neurons.
It was found that inhibition of CaMKII autophosphorylation re-
quired a higher concentration of curcumin than that was required
for substrate phosphorylation. Further, both the Ca2+-dependent
and Ca2+-independent activities of autophosphorylated CaMKII
were inhibited by curcumin I (1) (Table 2). The Ca2+/CaM-indepen-
standard deviation from a minimum of three experiments were calculated and are
shown in the table.
b
Indicates that the data shown is the same as in Table 1.
fect of curcumin on CaMKII in a more physiologically relevant
state, we have investigated the effect of curcumin on PSD and cyto-
solic fractions of CaMKII. Both the cytosolic and PSD fractions have
autophosphorylated and non-autophosphorylated forms of CaMKII
(Fig. 5), among which the autophosphorylated forms can exhibit
both Ca2+/CaM-dependent and Ca2+/CaM-independent activity.
The presence of autonomous CaMKII in cytosolic and PSD fractions
dent activitiy of T286D-
a
-CaMKII mutant, which is an autono-
were confirmed by immunoblot probed with anti-phospho-Thr286
-
mously active, autophosphorylation mimicking mutant form, was
also inhibited by curcumin I (1) (Table 2). Since calmodulin is
not required for Ca2+-independent activity, we concluded that cur-
cumin I (1) must be binding directly to CaMKII, and subsequently
inferred that the inhibition of curcumin on CaMKII is independent
of calmodulin binding. It was also seen that the inhibition of the
Ca2+-dependent activity of CaMKII by curcumin prevailed even at
higher concentrations of calmodulin (data not shown).
CaMKII antibody (Fig. 5B). Curcumin I inhibited both Ca2+/CaM-
dependent and Ca2+/CaM-independent activities of CaMKII in PSD
(Table 3) and cytosol (Table 4). Autophosphorylation of CaMKII in
cytosolic and PSD fractions was also inhibited, but with slightly
higher IC50 values (Table 3 and 4).
3.3. Effect of phenylpyrazole-curcumin (4) on CaMKII
A heterocyclic analog of curcumin, named CNB-001, was shown
to facilitate the induction of LTP in rat hippocampal slices, to en-
hance memory in rat in an object recognition test and also to en-
hance CaMKII activity in vitro of primary cortical neurons and of
CaMKII activity in a commercial assay kit.38 CNB-001 has also been
reported to show broad neuroprotective activity based on cell cul-
ture studies where the cells were subjected to diverse toxic in-
sults.52 The ability of CNB-001 to cross blood brain barrier and
its bioavailability upon oral administration have also been estab-
lished.52 Another report indicated that curcumin blocked the corti-
costerone induced elevation of phospho-CaMKII and NR2B levels in
hippocampal neurons.37 In the light of these studies, we investi-
gated the effect of phenylpyrazole-curcumin (4) on CaMKII. The
phenylpyrazole-curcumin (4) that we synthesized is structurally
identical to CNB-001, reported before.38,52 Contrary to the above
report, which indicates that CNB-001 activates CaMKII, we now
find that Ca2+-stimulated activity of CaMKII is inhibited by phenyl-
pyrazole-curcumin (4), based on our in vitro biochemical assay
(Table 5). Autophosphorylation, as well as Ca2+-dependent and
Ca2+-independent activities of autophosphorylated CaMKII, are
inhibited by phenylpyrazole-curcumin (4) (Table 5). Autophospho-
ryltion and Ca2+-dependent and Ca2+-independent activities of
CaMKII in cytosolic and in PSD fractions are also inhibited by phen-
ylpyrazole-curcumin (4) (Table 5).
3.2.3. Effect of curcumin I (1) on CaMKII in the cytosol and
postsynaptic density
CaMKII, being one of the most abundant proteins in the brain, is
found to be distributed both in the cytosol and localized at the
postsynaptic density (PSD) (Fig. 5A). In order to understand the ef-
Figure 4. Effect of curcumin I on Ca2+/CaM-dependent activitiy of
a-CaMKII. Dose
response plot as function of the concentration of curcumin I, is shown. Data from
four experiments is used to calculate the mean and standard deviation shown the
figure. The IC50 value for each experiment was calculated separately and the mean
and standard deviation were calculated and are shown in the tables.

M. Mayadevi et al. / Bioorg. Med. Chem. 20 (2012) 6040–6047
6045
Table 5
Inhibition of activity of CaMKII by phenylpyrazole-curcumin, pyrazole- curcumin and
isoxazole-curcumin
Type of CaMKII activity studied
IC50 lM
Phenylpyrazole-curcumin
Ca2+-dependent activity of CaMKII
37 6.40
79 9.37
54 8.5
111 11.49
94 27.78
96 22.16
80 34.2
103 6.98
62 12.95
68 26.93
Ca2+-dependent activity of autophosphorylated CaMKII
Ca2+-independent activity of autophosphorylated CaMKII
CaMKII autophosphorylation
Cytosol CaMKII Ca2+-dependent activity
Cytosol CaMKII Ca2+-independent activity
Cytosol CaMKII autophosphorylation
PSD CaMKII Ca2+-dependent activity
PSD CaMKII Ca2+-independent activity
PSD CaMKII autophosphorylation
Pyrazole-curcumin
Ca2+-dependent activity of CaMKII
1.49 0.46
6.3 2.80
0.29 0.05
6.5 3.38
Ca2+-dependent activity of autophosphorylated CaMKII
Ca2+-independent activity of autophosphorylated CaMKII
CaMKII autophosphorylation
Cytosol CaMKII Ca2+-dependent activity
Cytosol CaMKII Ca2+-independent activity
Cytosol CaMKII autophosphorylation
PSD CaMKII Ca2+-dependent activity
PSD CaMKII Ca2+-independent activity
PSD CaMKII autophosphorylation
4.4 1.90
0.44 0.023
16.32 1.63
3.6 1.08
0.34 0.33
7.58 2.43
Isoxazole-curcumin
Ca2+-dependent activity of CaMKII
11.37 4.08
24.44 0.96
0.68 0.04
18.01 1.61
28.9 1.79
13.68 2.75
17.10 4.84
22.87 6.06
18.88 4.59
22.79 4.23
Ca2+-dependent activity of autophosphorylated CaMKII
Ca2+-independent activity of autophosphorylated CaMKII
CaMKII autophosphorylation
Cytosol CaMKII Ca2+-dependent activity
Cytosol CaMKII Ca2+-independent activity
Cytosol CaMKII autophosphorylation
PSD CaMKII Ca2+-dependent activity
PSD CaMKII Ca2+-independent activity
PSD CaMKII autophosphorylation
The IC50 value for each experiment was analyzed separately and the mean and
standard deviation from a minimum of three experiments were calculated and are
shown in the table.
3.4. Effect of pyrazole-curcumin (5) on CaMKII
Figure 5. Western blot showing the presence of A: CaMKII in PSD (40
l
g) and
g)
lg) of rat brain. MW represents molecular weight
g) of rat brain and B: Thr286-phospho-CaMKII in PSD (40
l
Since phenylpyrazole-curcumin (4) inhibited CaMKII activity,
we next decided to investigate whether the simpler, unsubstituted,
pyrazole-curcumin (5) would have any effect on CaMKII. This was
investigated because the effect of pyrazole-curcumin (5) on CaMKII
has not been hitherto studied. Further, compared to phenylpyra-
zole-curcumin (4), pyrazole-curcumin (5) would be more similar
in structure to natural curcumin-1 (1), as pyrazole-curcumin (5)
lacks the bulky phenyl group. In addition, pyrazole-curcumin (5)
is a hydrogen bond donor through its NH group and would be less
hydrophobic (Fig. 6). We find that the Ca2+-dependent activity of
CaMKII is inhibited by pyrazole-curcumin (5) with a much lower
IC50 (Table 5) compared to phenylpyrazole-curcumin. Autophos-
phorylation of CaMKII is also inhibited by pyrazole-curcumin (5),
but with a higher IC50 value compared to that of the Ca2+
stimulated substrate phosphorylation activity (Table 5) of the
cytosolic fractions (30
l
and cytosolic fractions (30
marker.
Table 3
Inhibiton of activity of CaMKII in PSD by natural curcumin Ia
Type of CaMKII activity studied
IC50 lM
Ca2+-dependent activity of CaMKII in PSD
Ca2+-independent activity of CaMKII in PSD
Autophosphorylation of CaMKII in PSD
80 15.6
33 6.3
69 3.3
a
The IC50 value for each experiment was analyzed separately and the mean and
standard deviation from a minimum of three experiments were calculated and are
shown in the table.
non-autophosphorylated enzyme. Both Ca2+-dependent and Ca2+
-
independent activities of autophosphorylated CaMKII are inhibited
by pyrazole-curcumin (5). Interestingly, pyrazole-curcumin (5) is a
more potent inhibitor of the Ca2+-independent activity than that of
the Ca2+-dependent activity (Table 5). Autophosphorylation and
Ca2+-dependent substrate phosphorylation activity are both
dependent on the presence of Ca2+/calcmodulin and thus are likely
to arise from a similar conformational state of the enzyme. The
Ca2+-independent activity arises from the enzyme that is not
having significant amounts of bound Ca2+/calmodulin and hence,
may be in a different conformational state, which could be more
sensitive to pyrazole-curcumin. Pyrazole-curcumin inhibited
Table 4
Inhibiton of activity of CaMKII in cytosol by natural curcumin Ia
Type of CaMKII activity studied
IC50 lM
Ca2+-dependent activity of CaMKII in cytosol
Ca2+-independent activity of CaMKII in cytosol
Autophosphorylation of CaMKII in cytosol
112 22.8
64 15.9
143 27.9
a
The IC50 value for each experiment was analyzed separately and the mean and
standard deviation from a minimum of three experiments were calculated and are
shown in the table.

6046
M. Mayadevi et al. / Bioorg. Med. Chem. 20 (2012) 6040–6047
Figure 6. Representation of hydrogen bonding possible in curcumin I (1) and its heterocyclic derivatives (4-6).
autophosphorylation of CaMKII in PSD and cytosolic fractions.
Both Ca2+-dependent and Ca2+-independent activities of CaMKII
in PSD and cytosolic fractions are inhibited by pyrazole-curcumin
(Table 5).
4. Concluding remarks
CaMKII is a downstream component of the neuronal glutamate
signaling pathway and is deemed to be involved in the route to
glutamate-induced excitotoxicity. Hence acute inhibition of CaM-
KII may be a potential strategy for providing neuroprotection as
shown by two independent groups using the CaMKII inhibitor
tat-CN21 peptide.21,53 We now report that curcumin and its ana-
logues show inhibition of the Ca2+-dependent and Ca2+-indepen-
dent activities of CaMKII in vitro. Among the three cyclic
derivatives, pyrazole-curcumin (5) is most effective as an inhibitor
of CaMKII, even better than the parent curcumin I. Further, pyra-
zole-curcumin (5) is more effective on Ca2+-independent ‘‘autono-
mous’’ activity in the case of purified CaMKII and also in PSD and
cytosolic CaMKII fractions. Thus, pyrazole-curcumin (5) as well as
natural curcumin I (1) could offer neuroprotective action in the
excitotoxic cascade by inhibiting CaMKII activity. On the basis of
the above observations, it will be interesting to study further the
CaMKII signaling pathways that are altered by curcumin.
3.5. Effect of isoxazole-curcumin (6) on CaMKII
Subsequent to the investigations described above, we next
examined the effect of isoxazole-curcumin (6) on CaMKII as isoxaz-
ole ring is isosteric with pyrazole ring. This study showed that
Ca2+-dependent activity of CaMKII is inhibited by isoxazole-curcu-
min (6) with an IC50 value which is higher than that is observed for
pyrazole-curcumin (5) (Table 5). Autophosphorylation, Ca2+
-
dependent and Ca2+-independent activities of autophosphorylated
CaMKII are inhibited by isoxazole-curcumin (6) (Table 5). We find
that, even in the case of isoxazole-curcumin, the Ca2+-independent
activity of autophosphorylated CaMKII is more sensitive compared
to its Ca2+-dependent activities similar to pyrazole-curcumin. Isox-
azole-curcumin (6) inhibited the autophosphorylation of CaMKII in
PSD and cytosolic fractions and also the Ca2+-dependent and Ca2+
independent activities as well (Table 5).
-
Acknowledgements
The structure of curcumin is characterized by two ortho-meth-
oxyphenolic rings connected by linear C7 carbon chain bearing
three trans carbon–carbon double bonds, an oxo function, an enol
moiety and an enone group. It has been suggested that the termini
are hydrophilic whereas the middle carbon chain portion is very
hydrophobic, an observation that would substantiate the extreme
water insolubility of curcuminoids. Spectroscopic and crystallo-
graphic evidence have shown the central 1,3-diketone unit exist
exclusively as an intramolecularly hydrogen bonded 1,3-keto-enol
unit which would thus provide much rigidity to the overall linear
structure of the molecule. Such a rigidity can further be bolstered
by converting the central 1,3-diketone ¡ 1,3-keto-enol unit into
a pyrazole or an isoxazole ring. This heterocyclization of curcumin
I to pyrazole-curcumin would also alter the hydrogen bonding abil-
ity of this central part of the curcumin structure. Thus, in curcumin,
the central intramolecularly hydrogen bonded 1,3-keto-enol could
form one hydrogen bond through the enol oxygen, acting as an
hydrogen bond acceptor, to an external protein molecule (Fig. 6).
Such would be the case in phenylpyrazole-curcumin, and in isox-
azole-curcumin. In contrast, pyrazole-curcumin is capable of form-
ing two hydrogen bonds externally. These possibilities are
schematically shown in Figure 6, which imply that, other parame-
ters that affect molecular recognition being the same, the pyrazole-
curcumin could form a better hydrogen bonding. The higher inhib-
itory effect of pyrazole-curcumin may partly be ascribed to its abil-
ity to form better binding to CaMKII.
Financial support as intramural funds from RGCB and from
Department of Biotechnology, Government of India is gratefully
acknowledged. DRS thanks University of Kerala and VSK thanks
Kerala State Council for Science, Technology and Environment,
Government of Kerala for fellowships.
References and notes
1. Tobimatsu, T.; Fujisawa, H. J. Biol. Chem. 1989, 264, 17907.
2. Bennett, M. K.; Erondu, N. E.; Kennedy, M. B. J. Biol. Chem. 1983, 258, 12735.
3. Lisman, J. E.; Schulman, H.; Cline, H. Nat. Rev. Neurosci. 2002, 3, 175.
4. Lee, S. R.; Lozoya, Y. E.; Szatmari, E. M.; Yasuda, R. Nature 2009, 458, 299.
5. De Koninck, P.; Schulman, H. Science 1998, 279, 227.
6. Rosenberg, O. S.; Deindl, S.; Sung, R. J.; Nairn, A. C.; Kuriyan, J. Cell 2005, 123,
849.
7. Hudmon, A.; Schulman, H. Ann. Rev. Biochem. 2002, 71, 473.
8. Hudmon, A.; Schulman, H. Biochem. J. 2002, 364, 593.
9. Kuret, J.; Schulman, H. J. Biol. Chem. 1985, 260, 6427.
10. Colbran, R. J. Biochem. J. 2004, 378, 1.
11. Hajimohammadreza, I.; Probert, A. W.; Coughenour, L. L.; Borosky, S. A.; Boxer,
P. A.; Marcoux, F. W.; Wang, K. K. W. J. Neurosci. 1995, 15, 4093.
12. Bok, J.; Wang, Q.; Huang, J.; Green, S. H. Mol. Cell. Neurosci. 2007, 36, 13.
13. Tokumitsu, H.; Chijiwa, T.; Hagiwara, M.; Mizutani, A.; Terasawa, M.; Hidaka,
H. J. Biol. Chem. 1990, 265, 4315.
14. Sumi, M.; Kiuchi, K.; Ishikawa, T.; Ishi, A.; Hagiwara, M.; Nagatsu, T.; Hidaka, H.
Biochem. Biophys. Res. Commun. 1991, 181, 968.
15. Chang, B. H.; Mukherji, S.; Soderling, T. R. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
10890.
16. Chang, B. H.; Mukherji, S.; Soderling, T. R. Neuroscience 2001, 102, 767.

M. Mayadevi et al. / Bioorg. Med. Chem. 20 (2012) 6040–6047
6047
17. Vest, R. S.; Davies, K. D.; O’Leary, H.; Port, J. D.; Bayer, K. U. Mol. Biol. Cell 2007,
18, 5024.
18. Lepicard, E. M.; Mizuno, K.; Antunes-Martins, A.; Von Hertzen, L. S.; Giese, K. P.
Eur. J. Neurosci. 2006, 23, 3063.
19. Radwanska, K.; Tudor Jones, A. A.; Mizuno, K.; Pereira, G. S.; Lucchesi, W.;
Alfano, I. A.; Lach, I.; Kaczmarek, I.; Knapp, S.; Giese, K. P. Genes Brain Behav.
2010, 9, 648.
36. Ali, E. H. A.; Arafa, N. M. S. Fitoterapia 2011, 82, 601.
37. Xu, Y.; Lin, D.; Li, S.; Li, G.; Shyamala, S. G.; Barish, P. A.; Vernon, M. M.; Pan, J.;
Ogle, W. O. Neuropharmacology 2009, 57, 463.
38. Maher, P.; Akaishi, T.; Schubert, D.; Abe, K. Neurobiol. Aging 2010, 31, 706.
39. Babu, K. V. D.; Rajasekharan, K. N. Org. Prep. Procedures Intl. 1994, 26, 674.
40. Mishra, S.; Karmodiya, K.; Surolia, N.; Surolia, A. Bioorg. Med. Chem. 2008, 16,
2894.
20. Lucchesi, W.; Mizuno, K.; Giese, K. P. Brain Res. Bull. 2011, 85, 2.
21. Vest, R. S.; O’Leary, H.; Coultrap, S. J.; Kindy, M. S.; Bayer, K. U. J. Biol. Chem.
2010, 285, 20675.
22. Chattopadhyay, I.; Biswas, K.; Bandyopadhyay, U.; Banerjee, R. K. Curr. Sci.
2004, 87, 44.
23. Jain, S.; Shrivastava, S.; Nayak, S.; Sumbhate, S. Pharmacognosy Rev. 2007, 1,
119.
24. Balasubramanian, K. J. Agric. Food Chem. 2006, 54, 3512.
25. Jayaprakasha, G. K.; Rao, L. J.; Sakariah, K. K. Food Chem. 2006, 98, 720.
26. Frautschy, S. A.; Hu, W.; Kim, P.; Miller, S. A.; Chu, T.; Harris-White, M. E.; Cole,
G. M. Neurobiol. Aging 2001, 22, 993.
27. Yang, F.; Lim, G. P.; Begum, A. N.; Ubeda, O. J.; Simmons, M. R.; Ambegaokar, S.
S.; Chen, P.; Kayed, R.; Glabe, C. G.; Frautschy, S. A.; Cole, G. M. J. Biol. Chem.
2005, 280, 5892.
28. Jurenka, J. S. Altern. Med. Rev. 2009, 14, 141.
29. Mohan, R.; Sivak, J.; Ashton, P.; Russo, L. A.; Pham, B. Q.; Kasahara, N.; Raizman,
M. B.; Fini, M. E. J. Biol. Chem. 2000, 275, 10405.
30. Ruby, A. J.; Kuttan, G.; Babu, K. D.; Rajasekharan, K. N.; Kuttan, R. Cancer Lett.
1995, 94, 79.
31. Seo, K.-I.; Choi, M.-S.; Jung, U. J.; Kim, H-J; Yeo, J.; Jeon, S-M; Lee, M-K Mol. Nut.
Food Res. 2010, 52, 995.
32. Wang, M. S.; Boddapati, S.; Emadi, S.; Sierks, M. R. BMC Neurosci. 2010, 11, 57.
33. Sun, A. Y.; Wang, Q.; Simonyi, A.; Sun, G. Y. Neuromolecular Med. 2008, 10, 259.
34. Chen, H.-W.; Huang, H.-C. Br. J. Pharmacol. 1998, 1029, 124.
35. Lim, G. P.; Chu, T.; Yang, F.; Beech, W.; Frautschy, S. A.; Cole, G. M. J. Neurosci.
2001, 21, 8370.
41. Flynn, D. F.; Belliotti, T. R.; Boctor, A. M.; Cornnor, D. T.; Kostlan, C. R.; Nies, D.
E.; Ortwine, D. F.; Schrier, D. J.; Sircar, J. C. J. Med. Chem. 1991, 34, 518.
42. Ishida, J.; Ohtsu, H.; Tachibana, Y.; Nakanishi, Y.; Bastow, K. F.; Nagai, M.;
Wang, H. K.; Itokawa, H.; Lee, K. H. Bioorg. Med. Chem. 2002, 10, 3481.
43. Sui, Z.; Salto, R.; Li, J.; Craik, C.; de Montellano, P. R. O. Bioorg. Med. Chem. 1993,
1, 415.
44. Pradeep, K. K.; Cheriyan, J.; Suma Priya, S.; Rajeevkumar, R.; Mayadevi, M.;
Praseeda, M.; Omkumar, R. V. Biochem. J. 2009, 419, 123.
45. Laemmli, U. K. Nature 1970, 227, 680.
46. Praseeda, M.; Pradeep, K. K.; Krupa, A.; Sri Krishna, S.; Leena, S.; Rajeev Kumar,
R.; Cheriyan, J.; Mayadevi, M.; Srinivasan, N.; Omkumar, R. V. Biochem. J. 2004,
378, 391.
47. Towbin, H.; Staehelin, T.; Gordon, J. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 4350.
48. Anand, P.; Thomas, S. G.; Kunnumakkara, A. B.; Sundaram, C.; Harikumar, K. B.;
Sung, B.; Tharakan, S. T.; Misra, K. P.; Priyadarsini, I. K.; Rajasekharan, K. N.;
Aggarwal, B. B. Biochem. Pharmacol. 2008, 76, 1590.
49. Mayadevi, M.; Praseeda, M.; Kumar, K. S.; Omkumar, R. V. Biochim. Biophys. Acta
2002, 1598, 40.
50. Suenaga, T.; Morinobu, S.; Kawano, K.; Sawada, T. Int. J. Neuropsychopharmacol.
2004, 7, 299.
51. Atkins, C. M.; Chen, S.; Alonso, O. F.; Dietrich, W.; Hu, B. R. J. Cerebral Blood
Metabol. 2006, 26, 1507.
52. Liu, Y.; Dargusch, R.; Maher, P.; Schubert, D. J. Neurochem. 2008, 105, 1336.
53. Ashpole, N. M.; Hudmon, A. Mol. Cell. Neurosci. 2011, 46, 720.