Choline Kinase Inhibition and Docking Studies of a Series of 6-

(benzylthio)-9H-purin-9-yl-pyridinium derivatives

Belén Rubio-Ruiz1 Pablo Ríos-Marco2 María Paz Carrasco-Jiménez2 Antonio

Espinosa1 Ramon Hurtado-Guerrero3 Carmen Marco2 Ana Conejo-García1 and

Antonio Entrena1

✉Ana Conejo aconejo@ugr.esAntonio Entrenaaentrena@ugr.es

1 Department of Pharmaceutical and Organic ChemistryFaculty of Pharmacy, University of Granada 18071 Granada, Spain

2 Department of Biochemistry and Molecular Biology IFaculty of Sciences, University of Granada18071 Granada, Spain

3 Institute for Biocomputation and Physics of Complex Systems (BIFI)University of Zaragoza, 50018 Zaragoza, Spain.

1

Abstract

Human choline kinase (ChoK) is a well validated target for the treatment of cancer. In the last two

decades, many ChoK inhibitors have been developed and one of them is currently under evaluation

in clinical trials. In this paper a series of 6-(benzylthio)-9H-purin-9-yl-pyridinium derivatives were

evaluated as ChoK inhibitors, and their effects on cell proliferation were also investigated in the

human hepatoma HepG2 cell line. The most potent inhibitor against purified ChoK-α1 presents an

IC50 value of 0.4 μM. The biological data and the docking studies described here, support that the 4-

(dimethylamino)pyridinium cationic head and a small linker (benzene or biphenyl) are the essential

structural parameters for ChoK inhibition of the tested compounds.

Keywords: choline kinase; inhibitors; pyridinium compounds; antiproliferative agents;

lipophilicity.

2

Introduction

Choline kinase (ChoK) is an essential enzyme for the biosynthesis of phosphatidylcholine and

phosphatidylethanolamine, the most abundant phospholipids in eukaryotic membranes and precursors

of second messengers that can modulate growth or survival pathways (Gibellini and Smith, 2010).

This cytosolic enzyme phosphorylates both choline (Cho) and ethanolamine (Etn) in the presence of

ATP and Mg2+ to render phosphocholine (PCho) and phosphoethanolamine (PEtn) respectively, which

constitutes the first step of the Kennedy Pathway. ChoK is encoded by two genes, which express three

isoforms ChoK1, ChoK2 and ChoKβ that are enzymatically active as homodimeric or

heterodimeric forms (Aoyama et al., 2004). Homodimeric forms of both ChoKα1 and ChoKα2

display a dual choline and ethanolamine kinase activity, whereas the ChoKβ homodimer

predominantly has ethanolamine kinase activity, and the ChoKα–ChoKβ heterodimer has an

intermediate substrate specificity (Gallego-Ortega et al., 2009).

During the tumour formation the phospholipid metabolism is altered leading to high PCho

levels associated with an increased expression and activity of ChoK (Glunde et al., 2011).

Overexpression of ChoK predominantly as ChoK1 has been reported in numerous tumour types

including breast, liver, lung, colorectal, ovarian and prostate (Ling et al., 2015; Granata et al.,

2015). In addition, ChoKα has been identified as a downstream mediator of EGFR/c-Src signaling

in breast cancer cell lines (Miyake and Parsons, 2012) and its inhibition triggers apoptosis in cancer

cells by inducing exacerbated endoplasmic reticulum stress (Sanchez-Lopez et al., 2013). Due to its

vital role in tumour formation, ChoKα has been proposed and validated as a molecular target for the

development of novel cancer therapeutic agents (Aoyama et al., 2004; Glunde et al., 2011). In this

respect, small interfering RNAs (siRNA) and chemically synthesised ChoK inhibitors have proven

to be effective antitumor drugs in vitro and in vivo (Glunde et al., 2005; Campos et al., 2003;

Campos et al., 2006; Báñez-Coronel et al., 2008; Arlauckas et al., 2016; Zech et al., 2016). Finally,

a recent study suggests the critical role of ChoKβ, in concert with ChoKα, as a druggable target in

3

carcinogenesis (Chang et al., 2016).”

We have previously reported the rational design, synthesis and biological evaluation of a

series of non-symmetrical ChoK inhibitors possessing an adenine moiety (A or B), a 4-substituted

pyridinium ring (D or E), and a linker (F, G, H or I) that connects both fragments (Figure 1,

compounds 1-13) (Rubio-Ruiz et al., 2012). In compounds 1-8 (Family A), the linker is connected

to the adenine N-9 nitrogen atom, while in compounds 9-13 (Family B) this connection is through

the adenine N-3 nitrogen atom. The compounds from these libraries were biologically evaluated,

and showed moderate potency as ChoK inhibitors and antiproliferative agents against human

hepatoma HepG2 cell line (Rubio-Ruiz et al., 2012). Compounds 14-21 (Family C) were later

designed with the objective of improving the antiproliferative activity of previous molecules by

enhancing the lipophilicity (Rubio-Ruiz et al., 2013). In this sense, the adenine 6-NH2 amino group

of compounds 1-8 was replaced by a 6-benzylthio fragment. To assess the influence of lipophilicity

on biological activity, the antiproliferative effect of compounds 1-8 and 14-21 were evaluated

against human cervical carcinoma HeLa cell line. The results showed that compounds 14-21 are

better antiproliferative agents than compounds 1-8, and support the notion that the increased

lipophilicity brought about by the benzylthio group favourably affects the antitumor activity

(Rubio-Ruiz et al., 2013).

Figure 1

In this paper, we aim to gain insights into the ChoK inhibitory activity of compounds 14-21

not previously explored. Utilising two different in vitro inhibition assays and also docking studies,

we have assessed how the presence of a benzylthio fragment in position 6 of the adenine affects the

ChoK inhibitory activity of these structures.

Material and methods

4

Cell culture

Human hepatoma HepG2 cell line was obtained from The European Collection of Animal Cell

Cultures (Salisbury, UK) and cultured as described by Rubio-Ruiz et al (Rubio-Ruiz et al., 2012).

WST-1 Cell Viability Assay

HepG2 cells were seeded and treated as previously reported (Rubio-Ruiz et al., 2012). DMSO-

treated cells were used as control and never exceeded 0.3% concentration. To measure the

metabolic activity of cells, 10 μl (per 100 μl medium) of Cell Proliferation Reagent WST-1 was

added and incubated for 2-4 h at 37 ºC and 5% CO2. Each sample was analysed using a microplate

ELISA reader Bio-Tex-Instruments ELx800 (Winooski, USA). The absorbance of the formazan

product was measured at 450 nm. At least three experiments conducted in triplicate were performed

in all assays. The IC50 was determined from the dose-response curves according to the inhibition

ratio for each concentration by using the ED50plus v1.0 software.

Determination of choline kinase activity

In the present work, the inhibitory activity of compounds 14-21 was determined using ChoK

isolated from the cytosolic fraction of HepG2 cells (Rubio-Ruiz et al., 2012) and ChoK-α1 that it

was cloned and purified as previously described (Sahún-Roncero, Rubio-Ruiz, Saladino et al.,

2013).

The first assay relies on the phosphorylation of [methyl-14C]choline by the ChoK enzyme activity.

Due to its sensitivity this is one of the most appropriate methods to determine this enzyme activity

in cell extracts (Gallego-Ortega et al., 2009). Other analysis, such as the spectrophotometric assay

coupled to pyruvate kinase/lactate dehydrogenase requires a higher ChoK activity than the one

existing in the cytosolic fraction of the cell. Furthermore, when the effect of inhibitors on ChoK

activity has to be determined, the employment of assays coupled to other kinases must be avoided

since it cannot be neglected that these inhibitors can not only cause the inhibition of the ChoK

5

activity but also have the potential to simultaneously inhibit the kinase used for the coupled assay,

hence overestimating the inhibition of ChoK activity.

Choline kinase inhibition assay

ChoK activity in the cytosolic fraction of HepG2 cells was determined as previously reported

(Rubio-Ruiz et al., 2012). Similar protocol was used for purified ChoK-α1, with the difference that

20 ng of enzyme was added instead of cytosol and that the reaction lasted only for 10 min.

Inhibitors dissolved in DMSO were added to each enzymatic assay. DMSO-assays were always run

in parallel as a control. DMSO never exceeded a concentration of 0.2% to avoid unspecific ChoK

inhibition. At least three experiments conducted in triplicate were performed in all assays. The 50%

inhibitory concentrations (IC50 values) were determined from the % activity of the enzyme at

different concentrations of inhibitors by using a sigmoidal dose-response curve (ED50plus v1.0

software).

Docking Studies

Molecular modelling studies were performed using Sybyl program (SYBYL-X 2.0). Crystal

structure of human ChoKα1 in complex with compound 10 (PDB ID: 4BR3) was used for docking

studies (Sahún-Roncero, Rubio-Ruiz, Conejo-García et al., 2013). Protein structure was refined

using the Structure Preparation Tool module of Sybyl. Missing side chains of those residues

situated far away from the binding sites were added, and protein N-terminal and C-terminal were

fixed with ACE and NME, respectively. Hydrogens and charges were also added and protonation

type of Glu, Asp, Gln and Asp was analysed and fixed. Hydrogen orientations were also checked in

order to maintain intramolecular hydrogen bonds into the protein. Finally, compound 10 molecules

inserted into the ATP and Cho binding sites were carefully checked to assure the correction of these

molecules. Ligands were constructed from standard fragments of the Libraries of the Sybyl

program. As described previously (Conejo-García et al., 2003), a new type of atom was necessary

6

to define in order to build the molecules: N.ar4, the quaternary nitrogen atom of the pyridinium

fragments. Additional parameters were also developed from ab initio calculations to optimize the

geometry of these molecules (Conejo-García et al., 2003). Charges were calculated by means of

Gaussian Program (Gaussian 2004) and optimizations were tackled using the BFGS method.

The Surflex-Dock (Jain 2007) module implemented in the Sybyl program was used for docking

studies. Surflex Dock Protomol was prepared using the compound 10 inserted into the ChoK

binding site, with a threshold value of 0.5 and a Bloat of 0 Å. Surflex-Dock GeomX (SFXC)

protocol was used, the search grid was expanded in 5 Å, 50 additional starting conformation were

used for each molecule and 30 conformations per fragment. Results were analysed using Sybyl

program and the most stable pose for each molecule was chosen as the preferred one inside the

ChoK enzyme. Figures were built using the PyMOL program (PyMOL Molecular Graphics

System).

Results and Discussion

To determine ChoK inhibition for compounds 14-21 two different in vitro assays were

performed using human ChoK as a target (Table 1). Both tests enabled the evaluation of the affinity

of the compounds for ChoK without considering the possible uptake through cell plasma

membranes.

In order to compare inhibition against ChoK of compounds 14-21 (Family C) with the

previously published data for compounds 1-8 (Family A) (Rubio-Ruiz et al., 2012), we determined

the ChoK inhibitory activity using the cytosolic fraction isolated from HepG2 cells. As mentioned

previously, studies on the biological function of ChoK isoenzymes revealed that ChoK-α plays a

more prominent role in cancer development than ChoK-β (Gallego-Ortega et al., 2009). The mRNA

7

expression profile shows that the isoform ChoK-α is overexpressed in the HepG2 cell line (Ling et

al., 2015), providing an appropriate model to test the inhibitory activity. As previously described for

compounds 1-8 (Rubio-Ruiz et al., 2012), the results obtained from this assay show that the

presence of a dimethylamino group at position 4 of the pyridinium ring moiety leads to greater

ChoK inhibitory activity (compounds 14, 16, and 18 vs. compounds 15, 17, and 19, respectively). In

addition, it can be observed that ChoK inhibitory activity is favoured by smaller linkers as opposed

to compounds of Family A (Rubio-Ruiz et al., 2012). Under the same experimental conditions,

compound 14 which possesses benzene as a linker presents the lowest IC50 value of Family C (IC50

= 4.47 μM ± 0.51) while compound 7, possessing 1,4-diphenylbutane as a linker, is the most active

compound of Family A.

Table 1

Subsequently, the ChoK inhibitory activity of Family C was tested against the ChoK-α1

isoenzyme that was previously cloned and purified (Sahún-Roncero, Rubio-Ruiz, Saladino et al.,

2013). The structure-activity relationships established in the previous in vitro assay are maintained,

although all compounds show a better inhibitory profile with IC50 values below 20 µM with the

exception of compounds 17 and 21. Compound 14 is the most potent inhibitor of Family C against

purified ChoK-α1 (IC50 = 0.4 μM ± 0.03).

The observed differences in the ChoK inhibition data between both in vitro assays could be

a consequence of the lipophilicity of the inhibitors. Recent reports have demonstrated a compelling

correlation between lipophilicity and “pharmacological promiscuity” which describes a compound’s

pharmacological activity at multiple targets. These studies state that pronounced promiscuity is not

observed below a threshold cLogP value of 2 (Peters et al., 2009). The evaluation of the ChoK

inhibitory activity using HepG2 cells involves the disruption of the cell membrane to obtain the

cytosolic content, thus in the enzyme assay not only ChoK-α is present but also other related and

8

non-related enzymes, proteins, and subcellular membranes. In this assay, compounds with higher

cLogP values (i.e. compounds 17, 19-21; cLogP > 2) may target other enzymes and cell

components decreasing its effective concentration as its ChoK IC50 values show (Table 1).

Additionally, the antiproliferative activity of compounds 14-21 was evaluated on HepG2

cells after 24 hours treatment (Table 1). As previously described for HeLa cells, high cLogP values

favour the antiproliferative activity of these molecules (Rubio-Ruiz et al., 2013) which is an

important factor that affects compound's permeability to cross lipid membranes.

In order to understand the binding mode of compounds 14-21 to the enzyme, we performed

docking studies in the crystal structure of the ChoK-α1/10 complex (PDB ID: 4BR3, Sahún-

Roncero, Rubio-Ruiz, Conejo-García et al., 2013). Previous crystallographic studies of compounds

belonging to Family A and B demonstrated that compound 7 was able to occupy both Cho and ATP

binding sites simultaneously whereas compound 10 exclusively binds to the Cho-binding site. Due

to the steric bulk of the 6-benzylthio substituent and the absence of the H-bond donor group present

in the adenine 6-NH2 substituent, it is expected that these structures could be only inserted into the

Cho binding site, in a similar fashion to compound 10 observed in the ChoK-α1/10 crystal structure

(Figure S1). Indeed, our results show that compounds 14, 15, 16 and 18 may be inserted in the Cho

binding site similarly to compound 10. As observed in Figure 2 the 4-substituted-pyridinium

cationic head is stabilized by π-cation interactions with Tyr333, Tyr354, Tyr440, Trp420, Trp423

and Phe435. The smaller linker benzene present in compounds 14 (Figure 2A) and 15 (Figure 2B)

allows a deeper insertion of the 6-benzylthiopurine fragment into the Cho binding site in

comparison with the adenine moiety of compound 10, allowing additional interactions with the

enzyme: an additional H-bond between the purine N-1 atom and the OH residue of Tyr437; and a

stacking interactions between the benzylthio fragment and Ile433 side chain. These interactions also

favour the 4-(dimethylamino)pyridinium fragment of compound 14 to be closer to Trp420 than it is

in compound 10 allowing a stronger π-cation interaction. This fact may justify the slightly higher

9

ChoK inhibitory activity of compound 14 (IC50 = 4.47 μM, HepG2 cytosol) in relation to

compound 10 (IC50 = 6.21 μM, HepG2 cytosol). Compound 15 also shows good ChoK inhibition

(IC50: 7.91 μM, ChoK-α1 purified), however it is lower than compound 14 (IC50: 0.40 μM, ChoK-α1

purified). This difference may be the consequence of the presence of the 4-(pyrrolidin-1-

yl)pyridinium cationic head in compound 15. The higher steric bulk of the pyrrolidine ring prevents

the pyridinium moiety to be as close to Trp420 as it is in compound 14, giving a weaker π-cation

interaction with this amino acid.

Figure 2

As shown in Figure 2C, the obtained pose of compound 16 is quite similar to the crystal

structure of compound 10. Both molecules have a 1-(biphenyl-4-ylmethyl)-4-

(dimethylamino)pyridinium fragment but differ in the presence of the 6-benzylthiopurine moiety.

The purine fragments present in both structures are almost superimposed and situated outside of the

Cho binding site (Figure S2A), although the 6-benzylthio fragment of compound 16 is also

stabilized by an additional hydrophobic interaction with Gly119 (Figure 2C). This additional

interaction could explain the good ChoK inhibitory activity of compound 16 (IC50: 1.50 μM, ChoK-

α1 purified) which is slightly less potent that compound 14 since the 6-benzylthiopurine moiety is

outside of the Cho binding site and does not make the additional H-bond with Tyr437.

Compound 18 also contains a 4-(dimethylamino)pyridinium cationic head that is situated

into the Cho binding site and is stabilized by the π-cation interactions with Tyr333, Trp420 and

Trp423 (Figure 2D). Nevertheless, the 6-(benzylthio)purine moiety is away from the Cho binding

site probably due to the higher length of the 1,2-diphenylethane linker. The benzylthio fragment is

stabilized by a stacking interaction with the Gly119 (Figure S2B) but the general interactions of the

linker seems to be less potent than in compounds 14 and 16 since only the benzyl fragment

connected to the N-1 pyridinium moiety shows good π-cation interactions with Tyr354 and Tyr 440

explaining the lower ChoK inhibition of compound 18 (IC50 = 2.32 μM, ChoK-α1 purified).

10

Compound 20 shows a moderate ChoK inhibitory activity (IC50 = 18.83 μM, ChoK-α1

purified). This structure presents 4-(dimethylamino)pyridinium and 1,4-diphenylbutane moieties as

cationic head and linker respectively. The cationic head is inserted into the Cho binding site in a

similar manner to the previously described molecules. The 1-benzyl-4-(dimethylamino)pyridinium

is stabilized by π-cation interactions with Tyr333, Trp420, Trp423, Tyr354 and Tyr 440, but the rest

of the linker i.e. the 4-benzylethane fragment does not make the appropriate interactions with the

rest of the residues (Figure 3A). The length of the linker forces the 6-(benzylthio)purine to be away

from the enzyme (Figure S2C) and, consequently, does not interacts with the protein which also

may contribute to its low ChoK inhibitory activity.

Figure 3

Compounds 17, 19 and 21 present 4-(pyrrolidin-1-yl)pyridinium as cationic head and

possess longer linkers than compound 15. These structural features seem to be the reason for the

smaller activity of these molecules, since the cationic head does not interact with enzyme as 4-

(dimethylamino)pyridinium does. Furthermore, their 6-benzylthiopurine fragments are positioned

away from the protein.

As observed in Figure 3B, the obtained pose of compound 19 is similar to the one of

compound 18 (Figure 2D). The 4-(pyrrolidin-1-yl)pyridinium fragment is more distant from

Trp420 and, consequently, the π-cation interaction is lessened. In addition, the benzylthio fragment

is further from Gly119 in compound 19 making a small stacking interaction. Finally, compounds 17

and 21 show very high IC50 values (> 50 M, ChoK-1 purified). In both molecules, the 4-

(pyrrolidin-1-yl)pyridinium fragment adopts a similar disposition as compound 18 although their

benzylthio fragments are further from Gly119 (Figure 3B).

Conclusions

11

In summary we have used two different in vitro assays in order to investigate the ChoK

inhibitory activity of compounds 14-21. Their effects on cell proliferation were also investigated in

the HepG2 cell line. According to their activities, and supported by docking studies, the 4-

(dimethylamino)pyridinium cationic head and a small linker (benzene or biphenyl) are the essential

structural parameters for the ChoK inhibition of the tested compounds. Compound 14, that fits

these requirements, is the most potent inhibitor against purified ChoK-α1 with an IC50 value of 0.4

μM.

Acknowledgements

We thank the “Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía” (Excellence

Research Project no. P07-CTS-03210) and the “Ministerio de Ciencia e Innovación” (Project no.

SAF2009-11955) for the financial support. The award of grants from the “Ministerio de Educación”

to B.R-R. and P.R-M is gratefully acknowledged.

Conflicts of Interest

The authors declare that they have no conflicts of interest

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Table 1. cLogP[a], ChoK inhibition and antiproliferative effect against HepG2 cells of compounds

14-21[b]

Comp cLogP

IC50 ChoK

IC50 HepG2

HepG2 cytosol ChoK-α1 purified

14 0.1157 4.47 ± 0.51 0.40 ± 0.03 > 100

15 0.5248 32.19 ± 1.80 7.91 ± 0.94 > 100

16 1.6079 46.88 ± 1.34 1.50 ± 0.18 30.20 ± 1.15

17 2.2430 > 100 > 50 37.15 ± 1.17

18 1.6958 43.84 ± 1.20 2.32 ± 0.05 48.67 ± 3.30

19 2.1374 > 100 18.36 ± 0.65 44.67 ± 1.30

20 2.0462 > 100 18.83 ± 3.61 32.69 ± 6.91

16

21 2.4714 > 100 > 50 56.23 ± 1.09

[a] These results were previously published (Rubio-Ruiz et al., 2013). [b] Biological activities are

expressed as IC50 (µM) values ± SD.

Figure 1. Chemical structures of mono-quaternary pyridinium salts 1-21.

17

Figure 2. Superimposition of the obtained pose of compounds 14 (A, carbon atoms in green

colours), 15 (B, carbon atoms in magenta colours), 16 (C, carbon atoms in cyan colours) and 18 (D,

carbon atoms in white colours) with the molecule of compound 10 (carbon atoms in yellow colours)

inserted into the Cho binding site.

18

Figure 3. A) Superimposition of the obtained pose of compound 20 (carbon atoms in magenta

colours) with compound 10 (carbon atoms in yellow colours) inserted into the Cho binding site. B)

Superposition of the obtained pose of compounds 17 (carbon atoms in green colours), 19 (carbon

19

atoms in magenta colours) and 21 (carbon atoms in white colours) with compound 10 (carbon

atoms in yellow colours) inserted into the Cho binding site.

20