Structure guided design of potential inhibitors of human calcium-calmodulin dependent protein kinase IV containing pyrimidine scaffold

Article abstract of DOI:10.1016/j.bmcl.2015.12.098

Calmodulin dependent protein kinase IV (CAMKIV) belongs to the serine/threonine protein kinase family and considered as an encouraging target for the development of novel anticancer agents. The interaction and binding behavior of three designed inhibitors

Full text of DOI:10.1016/j.bmcl.2015.12.098

Accepted Manuscript
Structure guided design of potential inhibitors of human calcium-calmodulin
dependent protein kinase IV containing pyrimidine scaffold
Huma Naz, Ehtesham Jameel, Nasimul Hoda, Ashutosh Shandilya, Parvez
Khan, Asimul Islam, Faizan Ahmad, B. Jayaram, Md. Imtaiyaz Hassan
PII:
S0960-894X(15)30411-X
http://dx.doi.org/10.1016/j.bmcl.2015.12.098
BMCL 23459
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 September 2015
1 December 2015
29 December 2015
Please cite this article as: Naz, H., Jameel, E., Hoda, N., Shandilya, A., Khan, P., Islam, A., Ahmad, F., Jayaram,
B., Imtaiyaz Hassan, Md., Structure guided design of potential inhibitors of human calcium-calmodulin dependent
protein kinase IV containing pyrimidine scaffold, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://
dx.doi.org/10.1016/j.bmcl.2015.12.098
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Running head: Potential Inhibitors of CAMKIV
Structure guided design of potential inhibitors of human calcium-
calmodulin dependent protein kinase IV containing pyrimidine
scaffold
Huma Naza, Ehtesham Jameelb, Nasimul Hodac, *, Ashutosh Shandilyad,e , Parvez Khana, Asimul
Islama, Faizan Ahmada, B. Jayaramd,e, and Md. Imtaiyaz Hassana,*
a Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar,
New Delhi 110025, India
b Department of Chemistry, B.R. Ambedkar Bihar University, Muzaffarpur-842001, Bihar, India
c Department of Chemistry, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
d Supercomputing Facility for Bioinformatics & Computational Biology, e Department of
Chemistry, Indian Institute of Technology, Hauz Khas, New Delhi - 110016, India
*Corresponding Authors,
Md. Imtaiyaz Hassan, Ph.D.
(Assistant Professor)
Nasimul Hoda, Ph.D.
(Associate Professor)
Centre for Interdisciplinary Research
in Basic Sciences,
Department of Chemistry
Jamia Millia Islamia,
Jamia Millia Islamia,
Jamia Nagar, New Delhi 110025, India
Jamia Nagar, New Delhi 110025, India
E-mail: nhoda@jmi.ac.in
E-mail: mihassan@jmi.ac.in
1

Calmodulin dependent protein kinase IV (CAMKIV) belongs to the serine/threonine protein
kinase family and considered as an encouraging target for the development of novel anticancer
agents. The interaction and binding behavior of three designed inhibitors of human CAMKIV,
containing pyrimidine scaffold, was monitored by in vitro fluorescence titration and molecular
docking calculations under physiological condition. In silico docking studies were performed to
screen several compounds containing pyrimidine scaffold against CAMKIV. Molecular docking
calculation predicted the binding of these ligand in active-site cavity of the CAMKIV structure
correlating such interactions with a probable inhibition mechanism. Finally, three active
pyrimidine substituted compounds (molecule 1–3) have been successfully synthesized and
1
characterized by H & 13C NMR. Molecule 3 is showing very high binding-affinity for the
CAMKIV, with a binding constant of 2.2x108, M-1 ( 0.20). All three compounds are nontoxic to
HEK293 cells upto 50 µM. The cell proliferation inhibition study showed that the molecule 3 has
lowest IC50 value (46 1.08 µM). The theoretical and experimental observations are
significantly correlated. This study reveals some important observations to generate an improved
pyrimidine based compound that holds promise as a therapeutic agent for the treatment of cancer
and neurodegenerative diseases.
Keywords: Calcium-Calmodulin Dependent Protein Kinase IV; Drug Target; Molecular
Docking; Fluorescence Binding Study; High affinity ligands; Cancer and
Neurodegenerative diseases
2

Introduction
Calcium-calmodulin dependent kinase IV (CAMKIV) is a member of Ser/Thr family and
associated with numerous cellular activities such as cell signaling, cell cycling, apoptosis,
differentiation or proliferation, immune and inflammatory responses [1-4]. It is also involved in
the regulation of transcription factors through phosphorylation of the cAMP response element-
binding protein (CREB) [5-8]. Recently, it was shown that CAMKIV is increased and required
during Th17 cell differentiation, and its increased levels leads to the stimulation Th17-inducing
cytokines [9]. Extracellular stimuli like change in calcium concentration, inflammatory response
or hormonal stimuli cause the stimulation of CAMKIV [3, 10, 11]. On the other hand, over
expression or mutation in CAMKIV inhibits the autophosphorylation activity which leads to the
development of neurodegenerative diseases, and its transformation into oncogenic kinase
eventually increase the cancer risk [12]. Recent studies proven that CAMKIV is directly
associated with the hepatic [13] and epithelial ovarian cancers [14].
All these findings suggest that CAMKIV may be considered as a potential drug target for the
neurodegenerative diseases and cancer [15]. Despite of its potential role in the cellular
physiology and a close association with numerous diseases, a little attempt was taken for
designing any ligand/inhibitor of CAMKIV [16-18]. Corcoran and Means [19] have shown that
KN-93, an inhibitor of CAMKs had a dramatic effect on post-tetanic potentiation. Other CAMK
inhibitors such as KN-93 and KN-62 can induce the differentiation of multiple leukemic myeloid
cells (e.g. HL-60 and NB4) [20, 21]. Recent studies have clearly indicated that inhibiting
CAMKIV could help treat systemic lupus erythematosus [17, 22, 23]. Inhibition of CAMKIV in
MRL/lpr mice causes a significant suppression of nephritis and skin disease due to a remarkable
decrease in the expression of costimulatory molecules CD86 and CD80 on B cells, and
3

suppression of IFNγ and tumor necrosis factor α production [22]. Koga et al., [9] showed that
CAMKIV knockout or inhibition with a small molecule have decreased the severity disease.
Hence, development of few specific CAMKIV inhibitor and extending it to the development of
drug molecules is highly promising.
Recently, we have shown that curcumin, a natural anticancer agent, is showing a significant
binding-affinity to the CAMK4 [24]. To proceed further for designing a specific inhibitor of
CAMKIV, this is an attempt to synthesize few potential compounds that contain a pyrimidine
scaffold, with high binding-affinity. We constructed a ligand library of 100 compounds ends up
with three synthesizable lead molecule which are showing a significant binding affinity to the
CAMKIV, as confirmed by molecular docking and fluorescence binding studies. All three
1
molecules were successfully synthesized and characterized using H and 13C NMR and showing
a considerable binding affinity to the CAMKIV in vitro, that may be further utilized for the
design of potential drug molecules for the associated diseases.
Crystal structure of human CAMKIV (residues 15-340) has been deposited in the protein data
bank with PDB code 2W4O. However, there are many atoms are missing. Hence, we modeled
the structure of CAMKIV as described earlier [24]. We have successfully docked all three
molecules in the active-site cavity of the CAMKIV [25]. A reasonably high docking score was
obtained, indicating a strong binding affinity of these molecules with the CAMKIV. The
structure of CAMKIV is divided into five specific domains namely, autoinhibitory, nucleotide-
binding (305-321), serine/threonine phosphatase 2A (PP2A)-binding (306-323), CaM-binding
domain (322-341) (all four comprise the regulatory site) and the protein kinase domain (46-300),
4

the active site of the enzyme. Thr200, Ser12 andSer13 is the site of its phosphorylation [26].
Asp164 is an essential residue for the catalysis and acts as a proton acceptor. While, Lys75 is the
ATP-binding site [26].
Molecule 1 is present in the hydrophobic cavity of active-site. However, some portion of this
molecule is protruded out of the protein molecule (Figure 1A and 1B). Two hydrogen bonds are
formed by molecule 1 to the Glu168 and Asp185 of the CAMKIV accompanied by several van
der Waals interactions, clearly indicating the formation of a stable complex (Figure 1C). A
strong interaction of molecule 1 to the CAMKIV which have the most favorable binding energy
and clarifies the hydrogen-bonding and van der Waal's interactions with the important amino
acids, further suggest that molecule 1 as a potential ligand for the CAMKIV. This
pharmacological interaction is useful for better understanding of ligand binding mechanisms and
the potential use of molecule 1 as a therapeutic agent.
Table 1: Structures and free energy value of the synthesized compounds.
Molecule
R
Rʹ
∆G
Kcal/mol
-5.6
-7.38
-7.62
1
5
2
3
2
3
6
7
4
5

Molecular docking studies were carried out with a slightly bigger ligand (Molecule 2) to further
identify the binding mode inside the active site cavity of CAMKIV (Figure 2A). Molecule 2 is
expected to bind at the active site of CAMKIV (Figure 2B). However, it does not enters
6

completely to the cavity. Amino acids Glu168 and Lue52 of CAMKIV form H-bonds with the
molecule 2 supported by several hydrophobic interactions (Figure 2C). The docked ligand forms
a stable complex with human CAMKIV with a binding affinity (∆G in kcal/mol) value of −7.38
(Table 1). These preliminary results suggest that molecule 2 might exhibit inhibitory activity
against human CAMKIV, and may have further therapeutic application.
7

8

In order to get better ligand, we have decreased the size of ligand and designed molecule 3 for
further study (Figure 3A), which completely enters a deep inside the active site cavity of
CAMKIV (Figure 3B). This ligand is expected to bind efficiently to the active site residues of
protein with weak non-covalent interactions, most prominent of which are H-bonding, π–π
stacking and alkyl-π interactions (Figure 3B). Asp185 holds the molecule at the active site by
forming H-bond of 2.75 Å length with the nitrogen of pyrimidine (Figure 3C). Similarly,
Gly187 holds the molecule 3 by forming H-bond with the nitrogen of pyrimidine. However,
Val121 is forming H-bond with the oxygen atom of molecule 3. Several hydrophobic residues
also play role in stabilizing the ligand–macromolecular complex by forming an alkyl-π
interaction with central pyrimidine ring. Binding free energy (∆G in kcal/mol) was found to be
−7.62, as predicted by Pardock module of Sanjeevini, suggests a very high binding-affinity
between the ligand with CAMKIV. These results draw us to the conclusion that this molecule
might exhibit inhibitory activity against the CAMKIV. However biological tests need to be done
to validate the computational predictions.
9

Key steps in the synthesis of compounds shown in Table 1 involved nucleophilic aromatic
substitution of 4,6-dichloropyrimidine with various nucleophiles depicted in Scheme 1. Firstly,
4,6-dichloropyrimidine 1 was treated with different primary amine or phenol in presence of base
as (K2CO3 or DIEA) in DMF at room temperature to give compounds 2-3 as monosubstituted
pyrimidines in good yields. The formed mono-substituted pyrimidine was further treated with
different nucleophile such as amino-phenol with appropriate base (K2CO3, KOH or DIEA) in dry
10

DMF at higher temperature to give Molecule 1, 2 and 3 as disubstituted pyrimidines
(Scheme 1). The first as well as second substitution showed temperature dependent nucleophilic
aromatic substitution reactions in which the first chlorine was replaced at room temperature with
1 equivalent of nucleophile and 1-1.5 equivalents of appropriate base in DMF. While the second
chlorine replaced at higher temperature with 1-2.5 equivalents of nucleophile and 1.2-1.5
equivalents of appropriate base in dry DMF in dry nitrogen atmosphere. All the synthesized
compounds (2-3, molecule 1, 2, 3) were purified by column chromatography using EtOAc/Pet.
ether as eluent and characterized by various spectroscopic techniques.
X
X'
X
Cl
Cl
Cl
R1
R2
R1
(a)
(b)
N
N
N
N
N
N
1
2-3
4-6
Scheme 1: Reagents and conditions: (a) Appropriate base, DMF, r.t, 2-10hrs.
(b) Appropriate base, Dry DMF, Reflux, overnight. 1= 4,6-Dichloropyrimidine scaffold;
RR´= Aryl group; XX´= Different nucleophiles.
To prepare 2-(piperazin-1-yl)nicotinonitrile (C), a solution of Piperazine (3g), and K2CO3 (4.8 g,
1 eq.) in dioxane (15mL) was added 2-chloro-3-cyanopyridine (4.8 g, 1 eq.) and was stirred to
reflux at 100-110 °C for 4-5 hrs (Scheme 2). The resulting precipitate was filtered off and
concentrated at reduced pressure to obtain the crude product which was purified was silica gel
column chromatography MeOH:CHCl3 (5:95 v/v) to give compound C as yellow solid (3g, 46%)
[27].
11

A
B
C
Scheme 2: Reagents and conditions: (i). K2CO3, Dioxane, reflux, 4-5 hrs.
A= 2-Chloro-3-cyanopyridine, B= Piperazine.
To further validate in silico binding observations, we performed fluorescence binding studies of
synthesized molecules to the CAMKIV [28]. We have successfully cloned, expressed and
purified the kinase domain of CAMKIV (Method is described in the biology section). 2.5 mL
solution containing 4 µM of CAMKIV was titrated by the successive addition of 1.0 mM stock
solution of ligands and the concentrations were varied from 0 to 132 µM. Titrations were done
manually by using micro-injector. An excitation wavelength (Ex) of 280 nm was chosen in the
experiment and emission spectra were recorded in the range of 300 to 400 nm.
The effect of molecule 1, molecule 2 and molecule 3 on the fluorescence emission spectra of
CAMK4 (pH = 7.4) at 298 K are shown in Figures 4A, 5A and 6A, respectively. These data
were used for calculation of binding affinity of these molecules to the CAMKIV as provided in
the Table 2. As shown in these figures, the peak at fluorescence emission maxima of CAMKIV
is observed at around 343 nm, is gradually quenched by the increasing concentration of the
ligands, while the ligands has no intrinsic fluorescence in this range. These observations clearly
indicates that a strong interaction exist between designed molecules (Molecule 1, 2, and 3) and
CAMKIV. The relationship between decrease in fluorescence intensity and the concentration of
ligands can be described by the Stern–Volmer equation following Eq. (2), the apparent Ka is the
12

binding constant and n is the number of binding sites per protein molecule can be expressed for
ligand-CAMKIV system [29, 30].
Table 2: Binding affinity of designed compounds measured by fluorescence
*mean errors are given in the parenthesis for three measurements.
S.NO
Name of compound
Binding Constant*
No. of Binding site
1
2
3
Molecule 1
Molecule 2
Molecule 3
1.6x105, M-1 ( 0.12)
4.6x105, M-1 ( 0.16)
2.2x108, M-1 ( 0.20)
1.0
1.0
2.0
13

Figure 4A, 5A and 6A clearly indicate that all three molecules quench the fluorescence emission
of CAMKIV with higher binding affinity and experiences a static quenching process. The data of
fluorescence intensity at 343 nm were plotted against [ligand] to calculate the binding-affinity
and the number of binding sites (Figure 4B, 5B and 6B). It is interesting to note that molecule 1
and 2 are having binding affinity value as 1.6x105, M−1( 0.12) and 4.6x105, M−1 ( 0.16),
14

respectively. Both these molecules having a single binding site to the CAMKIV. However,
molecule 3 shows a significantly very high binding affinity for the CAMKIV (2.2x108, M−1
( 0.20)) and the number of binding site is two. All these observations clearly indicate that
molecule 3 is one of the very efficient ligand, and can be further exploited for the development
of novel pharmacophore for the CAMKIV associated diseases.
We started with a large number of diphenyl derivatives of pyrimidine. Out of 100 compounds,
we selected top 10 hits of docking results for further study. However, we did not successfully
synthesized all 10 compounds. Finally, three molecules were successfully synthesized and their
chemical analysis is reported in the chemistry section. These three molecules are showing a
considerable binding affinity to the CAMKIV.
MTT assay has gained high attention in cell proliferation studies. MTT is a tetrazolium salt,
which can be easily reduced to a colored formazan product with the help of reducing enzyme
15

present only in metabolically active or dividing cells [31]. The enzyme succinate dehydrogenase
and cytochrome oxidase present in mitochondria of active cells that help in the reduction of MTT
[32]. Thus under a particular condition reduction of MTT and subsequent formation of formazan
is proportional to the number of viable or active cells present in the medium [31, 32]. With the
help of MTT-assay we have analyzed the cytotoxicity and cell-proliferation inhibition activity of
designed inhibitors [33]. Comparison of the cell viability is shown in Figure 7A which clearly
revealed that molecule 3 nearly up to 50 µM, and molecules 1 and 2 are nearly up to 25 µM, was
non-toxic to normal cells. Above this concentration, the reference and the tested inhibitors both
became toxic to the normal cells.
We further performed the anti-cancerous activity of these inhibitors on human cancer cells and
found an appreciable activity against cancer cell line. From the percentage viability we have
calculated the inhibitory concentration (IC50) of each inhibitor. It is evident from Figure 7B that
these synthesized compounds display a dramatic anticancer activity for human hepatic cancer
cell-line. As shown in Table 3 for HuH7 cells, the molecule 1 and 2 show higher IC50 values as
compared to the molecule 3. Calculation shows that the molecule 3 inhibits the proliferation of
cancerous cells almost two times more than the molecule 1 and 2. These results are comparable
to curcumin, a natural anticancer compound used a positive control in this study. Our findings
also suggest that these inhibitors are selective and proven 8-10 times more active against
cancerous cells then normal one (Figure 7). Moreover, on HuH7 cells the molecule 3 shows the
better anti-proliferative activity in comparison to molecule 1 and 2, which was also
complimented by our binding and docking studies. The results of our in vitro and in silico studies
to a certain extent validate our strategies to design novel and potential inhibitors against
CAMKIV.
16

Table 3: IC50 (µM) values of designed inhibitors on HuH7 and HEK293 cells
Name of compound
IC50 (HuH7 cells) in
µM
IC50 (HEK293 cells) in
µM
>400
400 1.10
>400
aCurcumin
Molecule 1
Molecule 2
Molecule 3
48 1.11
116 1.12
104 1.77
46 1.08
>400
apositive control
In conclusion, CAMKIV is an important member of calcium-triggered signaling cascade,
involved in the regulation of transcription and microtubule dynamics. Its abnromal expression is
directly associated with varieties of cancer and neurodegenerative diseases. Three potential
ligands of CAMKIV were suggested by docking simulation studies, were successfully
synthesized and characterized. We further shown that all three molecules bind efficiently to the
active site of CAMKIV. Molecule 1 and 2 can bind to CAMKIV with stoichiometry ratio of 1:1,
however, molecule 3 in stoichiometry ratio of 2:1. The CAMKIV-ligand complex is stabilized by
several non-covalent interactions offered by residues in vicinity to the active site. We further
shown that molecule 3 is showing very good anticancerous activity on HuH7 cell line. No doubt,
these results are important in the field of pharmacy, pharmacology and biochemistry, and are
expected to shed some light on the binding property of CAMKIV with the ligands. These
findings may further explored for the drug design and discovery of potential drug molecules for
the diseases associated with the altered expression of CAMKIV.
Acknowledgements
The authors thank the Indian Council of Medical Research (ICMR), New Delhi for the financial
support to carry out this research work. Authors also thank Jamia Millia Islamia for providing
high speed server to the Central Instrumentation Facility. HN thanks to the ICMR for the award
17

of fellowship. CAMKIV gene was provided by DF/HCC DNA Resource Core, Harvard Medical
School. We sincerely thank Department of Science and Technology, Government of India for
FIST support [SR/FST/LS-541/2012].
Conflict of interest
Authors declare no conflict of interest regarding any financial and personal relationships with
other people or organizations that could inappropriately influence (bias) this work.
References
1.
Racioppi L.; Means A.R., Calcium/calmodulin-dependent kinase IV in immune and
inflammatory responses: novel routes for an ancient traveller. Trends Immunol. 2008, 29(12),
600-7.
2.
Ichinose K.; Rauen T.; Juang Y.T.; Kis-Toth K.; Mizui M.; Koga T.; Tsokos G.C.,
Cutting edge: Calcium/Calmodulin-dependent protein kinase type IV is essential for mesangial
cell proliferation and lupus nephritis. J Immunol. 2011, 187(11), 5500-4.
3.
Persaud S.J.; Liu B.; Sampaio H.B.; Jones P.M.; Muller D.S., Calcium/calmodulin-
dependent kinase IV controls glucose-induced Irs2 expression in mouse beta cells via activation
of cAMP response element-binding protein. Diabetologia. 2011, 54(5), 1109-20.
4.
Sugiyama Y.; Murao K.; Imachi H.; Sueyoshi N.; Ishida T.; Kameshita I.,
Calcium/calmodulin-dependent protein kinase IV involvement in the pathophysiology of
glucotoxicity in rat pancreatic beta-cells. Metabolism. 2011, 60(1), 145-53.
5.
Sun P.; Enslen H.; Myung P.S.; Maurer R.A., Differential activation of CREB by
Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a
site that negatively regulates activity. Genes Dev. 1994, 8(21), 2527-39.
6.
Liu B.; Barbosa-Sampaio H.; Jones P.M.; Persaud S.J.; Muller D.S.,
The
CaMK4/CREB/IRS-2 cascade stimulates proliferation and inhibits apoptosis of beta-cells. PloS
one. 2012, 7(9), e45711.
7.
Bell K.F.; Bent R.J.; Meese-Tamuri S.; Ali A.; Forder J.P.; Aarts M.M., Calmodulin
kinase IV-dependent CREB activation is required for neuroprotection via NMDA receptor-
PSD95 disruption. J Neurochem. 2013, 126(2), 274-87.
18

8.
Matthews R.P.; Guthrie C.R.; Wailes L.M.; Zhao X.; Means A.R.; McKnight G.S.,
Calcium/calmodulin-dependent protein kinase types II and IV differentially regulate CREB-
dependent gene expression. Mol Cell Biol. 1994, 14(9), 6107-16.
9.
Koga T.; Hedrich C.M.; Mizui M.; Yoshida N.; Otomo K.; Lieberman L.A.; Rauen T.;
Crispin J.C.; Tsokos G.C., CaMK4-dependent activation of AKT/mTOR and CREM-alpha
underlies autoimmunity-associated Th17 imbalance. J Clin Invest. 2014, 124(5), 2234-45.
10.
Atkins C.M.; Chen S.; Alonso O.F.; Dietrich W.D.; Hu B.R., Activation of
calcium/calmodulin-dependent protein kinases after traumatic brain injury. J Cereb Blood Flow
Metab. 2006, 26(12), 1507-18.
11.
Tokumitsu H.; Brickey D.A.; Glod J.; Hidaka H.; Sikela J.; Soderling T.R., Activation
mechanisms for Ca2+/calmodulin-dependent protein kinase IV. Identification of a brain CaM-
kinase IV kinase. J Biol Chem. 1994, 269(46), 28640-7.
12.
Lin F.; Marcelo K.L.; Rajapakshe K.; Coarfa C.; Dean A.; Wilganowski N.; Robinson H.;
Sevick E.; Bissig K.D.; Goldie L.C.; Means A.R.; York B., The CaMKK2/CaMKIV relay is an
essential regulator of hepatic cancer. Hepatology. 2015.
13.
Lin F.; Marcelo K.L.; Rajapakshe K.; Coarfa C.; Dean A.; Wilganowski N.; Robinson H.;
Sevick E.; Bissig K.D.; Goldie L.C.; Means A.R.; York B., The camKK2/camKIV relay is an
essential regulator of hepatic cancer. Hepatology. 2015, 62(2), 505-20.
14.
Takai N.; Miyazaki T.; Nishida M.; Nasu K.; Miyakawa I., Ca(2+)/calmodulin-
dependent protein kinase IV expression in epithelial ovarian cancer. Cancer Lett. 2002, 183(2),
185-93.
15.
Knapp S.; Sundstrom M., Recently targeted kinases and their inhibitors-the path to
clinical trials. Curr Opin Pharmacol. 2014, 17, 58-63.
16.
See V.; Boutillier A.L.; Bito H.; Loeffler J.P., Calcium/calmodulin-dependent protein
kinase type IV (CaMKIV) inhibits apoptosis induced by potassium deprivation in cerebellar
granule neurons. FASEB J. 2001, 15(1), 134-44.
17.
McCullough L.D.; Tarabishy S.; Liu L.; Benashski S.; Xu Y.; Ribar T.; Means A.; Li J.,
Inhibition of calcium/calmodulin-dependent protein kinase kinase beta and calcium/calmodulin-
dependent protein kinase IV is detrimental in cerebral ischemia. Stroke. 2013, 44(9), 2559-66.
18.
Liu G.; Voyno-Yasenetskaya T.A., Radixin stimulates Rac1 and Ca2+/calmodulin-
dependent kinase, CaMKII: cross-talk with Galpha13 signaling. J Biol Chem. 2005, 280(47),
39042-9.
19.
Corcoran E.E.; Means A.R., Defining Ca2+/calmodulin-dependent protein kinase
cascades in transcriptional regulation. J Biol Chem. 2001, 276(5), 2975-8.
20.
Yamamoto-Yamaguchi Y.; Okabe-Kado J.; Kasukabe T.; Honma Y., Induction of
differentiation of human myeloid leukemia cells by immunosuppressant macrolides (rapamycin
19

and FK506) and calcium/calmodulin-dependent kinase inhibitors. Exp Hematol. 2001, 29(5),
582-8.
21.
Si J.; Mueller L.; Schuler A.; Simon J.; Collins S.J., The retinoic acid receptor/CaMKII
interaction: pharmacologic inhibition of CaMKII enhances the differentiation of myeloid
leukemia cells. Blood Cells Mol Dis. 2007, 39(3), 307-15.
22.
Ichinose K.; Juang Y.T.; Crispin J.C.; Kis-Toth K.; Tsokos G.C., Suppression of
autoimmunity and organ pathology in lupus-prone mice upon inhibition of calcium/calmodulin-
dependent protein kinase type IV. Arthritis Rheum. 2011, 63(2), 523-9.
23.
Koga T.; Ichinose K.; Mizui M.; Crispin J.C.; Tsokos G.C., Calcium/calmodulin-
dependent protein kinase IV suppresses IL-2 production and regulatory T cell activity in lupus. J
Immunol. 2012, 189(7), 3490-6.
24.
Hoda N.; Naz H.; Jameel E.; Shandilya A.; Dey S.; Hassan M.I.; Ahmad F.; Jayaram B.,
Curcumin specifically binds to the human calcium-calmodulin-dependent protein kinase IV:
fluorescence and molecular dynamics simulation studies. J Biomol Struct Dyn. 2015, 1-13.
25.
The docking and scoring of ligands with CAMKIV protein was accomplished using
Pardock module of Sanjeevini drug design suite which is based on physico-chemical descriptors.
It is a rigid docking module which accomplishes a series of computational steps such as
preparation of reference protein complex and ligand in a force-field compatible manner as an
input file. Docking of ligand molecules at the active-site using all atom energy based Monte
Carlo algorithm, minimizes and scores the docked complex. The docked complexes are further
minimized using the parallel version of sander module of AMBER, predicted binding free energy
of docked poses are obtained using Bappl/BapplZ scoring function. The crystal structure of
complex, CAMKIV protein in complex with 4-Amino(sulfamoyl-phenylamino)-triazole-
carbothioic acid (2,6-difluoro-phenyl)-amide) (PDB ID 2W4O) was utilized as reference for
building new model and docking studies. The energy minimized structure of 100 ligands were
docked at the active site out which three potential ligands were chosen for further synthesis
(details are provided separately in the chemistry section) and binding studies.
26.
Krebs J., Calmodulin-dependent protein kinase IV: regulation of function and
expression. Biochim Biophys Acta. 1998, 1448(2), 183-9.
27.
Bhat H.R.; Pandey P.K.; Ghosh S.K.; Singh U.P., Development of 4-aminoquinoline-
1,3,5-triazine conjugates as potent antibacterial agent through facile synthetic route. Med Chem
Res. 2013, 22, 5056-65.
28.
Fluorescence measurements were carried out in the Jasco Spectrofluorimeter (Model FP-
6200) using 5 mm quartz cuvette. Experiment was carried out at 25 0.1 °C maintained by an
external thermostated water circulator. Stock solutions (5mg/ml) of synthesized compounds 1, 2,
3 were prepared in the DMSO and then diluted to 1 mg/ml in the 50 mM phosphate pH 7.4
buffer containing 150 mM NaCl (PBS). All compounds were further dialyzed in the PBS before
the experiment. Intrinsic fluorescence emission spectra of CAMKIV in the presence of different
concentrations of synthesized compounds were performed in the PBS at 25 °C. For this purpose
excitation wavelength was fixed at 280 nm and emission was at 300-400nm. The excitation and
20

emission slit widths (5 nm each) and scan rate (100 nm/min) were constantly maintained for all
experiments. A typical emission peak was observed at 343 nm. Final spectrum was collected
after deducting their corresponding blank. Three independent experiments were performed for
each compound and their average was taken for data analyses. A decrease in the fluorescence
intensity due to increasing concentrations of compounds were used as a parameter for calculating
the binding constant K and number of binding site n using modified Stern-Volmer equations with
slight modification.
log(Fo-F)/F = logKa + nlog[Q ]
(1)
Where, Fo is the fluorescence intensity of protein and F is the fluorescence intensity of ligand,
Ka is the binding constant and n is the number of binding sites, Q represents quenching constant.
For the ligand-protein complex, the values for Ka and n can be derived from the intercept and
slope.
29.
Garg A.; Manidhar D.M.; Gokara M.; Malleda C.; Suresh Reddy C.; Subramanyam R.,
Elucidation of the binding mechanism of coumarin derivatives with human serum albumin. PloS
one. 2013, 8(5), e63805.
30.
Agudelo D.; Bourassa P.; Bruneau J.; Berube G.; Asselin E.; Tajmir-Riahi H.A., Probing
the binding sites of antibiotic drugs doxorubicin and N-(trifluoroacetyl) doxorubicin with human
and bovine serum albumins. PloS one. 2012, 7(8), e43814.
31.
Mosmann T., Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J Immunol Methods. 1983, 65(1-2), 55-63.
32.
Slater T.F., Studies on a Succinate-Neotetrazolium Reductase System of Rat Liver. Ii.
Points of Coupling with the Respiratory Chain. Biochim Biophys Acta. 1963, 77, 365-82.
33.
Exponentially growing human heptoma cells (HuH7) and human embryonic kidney cells
(HEK293) were seeded in a 96-well micro titer plate (cell count of 7000 cells per well). For
checking cell proliferation, on the next day cells were treated with different concentrations (5
µM-150 µM) of inhibitors. Curcumin was used as a positive control. At the end of 48 hr
incubation, the mixture was removed from the cells and after washing with PBS, 100µl fresh
Dulbecco's modified Eagle's medium and 20 µl MTT (from 5mg/ml stock) was added to each
well. The plates were incubated further for 4-5 hr at 37°C. After the incubation period is over,
the bulk of the residual medium was removed using a multichannel pipette carefully without
disturbing the formazan crystals formed behind. After that 100 µl DMSO (dimethyl sulphoxide)
was added to each well for dissolving the formazan crystals. The plates were then agitated for
15-20 minutes on an orbital plate shaker, after which they were read immediately on Titerplate
reader (BioRad) at 570 nm. The relative cell viabilities (in percentage) were calculated with that
of the control by comparing the viability of the treated cells.
21

Figure Legends:
Figure 1: Binding of Compound 1 with the CAMK4. (A). Structure of 3-((6-(quinolin-8-
yloxy)pyrimidin-4-yl)oxy)aniline (Molecule 1) (B). Showing surface view of ligand
(stick model) present in the active site cavity of the CAMK4. (C). Showing
interaction of ligand (stick model) with the protein atoms (line model).
Figure 2: Binding of Compound 2 with the CAMK4. (A). Structure of 2-(4-(6-(quinolin-8-
yloxy)pyrimidin-4-yl)piperazin-1-yl)nicotinonitrile (Molecule 2) (B). Showing
surface view of ligand (stick model) present in the active site cavity of the CAMK4.
(B). Showing interaction of ligand (stick model) with the protein atoms (line model).
Figure 3: Binding of Compound 3 with the CAMK4. (A). Structure of N-(4-methoxyphenyl)-6-
(piperazin-1-yl)pyrimidin-4-amine (Molecule 3) (B). Showing surface view of ligand
(stick model) present in the active site cavity of the CAMK4. (C). Showing
interaction of ligand (stick model) with the protein atoms (line model).
Figure 4. Fluorescence binding study of compound 1 with the CAMK4. (A). Fluorescence
spectra of CAMKIV (4 µM) in the increasing concentrations of ligand 8, 16, 24, 32,
40, 48, 56, 64, 72, 80, 88, 96 µM (from top to bottom) at pH 7.4. The protein was
excited at 280nm and emission spectra was collected in the range of 300-400 nm) (B).
Modified Stern–Volmer plot for tryptophan quenching of CAMKIV by compound 1
used for the calculation of binding affinity.
Figure 5. Fluorescence binding study of compound 2 with the CAMK4. (A). Fluorescence
spectra of CAMKIV (4 µM) in the increasing concentrations of ligand 0, 6, 12, 18,
22

24, 30, 36, 42, 48, 54 µM (from top to bottom) at pH 7.4. The protein was excited at
280nm and emission spectra was collected in the range of 300-400nm) (B). Modified
Stern–Volmer plot for tryptophan quenching of CAMKIV by compound 2 used for
the calculation of binding affinity.
Figure 6. Fluorescence binding study of compound 3 with the CAMK4. (A). Fluorescence
spectra of CAMKIV (4 µM) in the increasing concentrations of ligand 0, 20, 26, 32,
38, 44, 50, 56, 62, 68, 74, 80, 86, 92, 98, 104, 110, 116, 120, 126, 132 µM (from top
to bottom) at pH 7.4. The protein was excited at 280nm and emission spectra was
collected in the range of 300-400nm) (B). Modified Stern–Volmer plot for tryptophan
quenching of CAMKIV by compound 3 used for the calculation of binding affinity.
Figure 7: (A). Cytotoxicity results of synthesized inhibitors (molecule 1, 2, and 3) on the
viability of HEK293 cells. A total of 7000 cells in triplicates were exposed to the
indicated concentration of inhibitors for 48 h and subjected to MTT assay. (B). Effect
of synthesized inhibitors (molecule 1, 2, and 3) on the proliferation of HuH7 cells. A
total of 7000 cells in triplicates were exposed to the indicated concentrations of
curcumin and inhibitors for 48 h and subjected to MTT assay. Data represent three
independent sets of experiments and results are shown as the mean S.D. Curcumin
is used as a positive control.
23

*Graphical Abstract (for review)
Structure Guided Design of Potential Ligand of Human Calcium-Calmodulin Dependent Protein Kinase IV Containing Pyrimidine Scaffold
Huma Naz, Ehtesham Jameel, Nasimul Hoda, Ashutosh Shandilya , B. Jayaram, Faizan Ahmad, Asimul Islam, and Md. Imtaiyaz Hassan*
Highlights
•Three potential ligands were docked in the active site cavity of CAMKIV.
•Several non-covalent interactions are formed between ligands and CAMKIV.
•All three ligands were synthesized and characterized successfully.
•Molecule 3 is showing very high affinity for the CAMKIV in the nm range.
Molecule 3 in the active site cavity of CAMKIV Showing various non-covalent interaction