Research Article

In silico analysis of hexokinase- I and II as potential drug targets in cancer

Sudhir Ranjan Bhoi, Nihar Ranjan Panda, Rakesh M. Rawal, Mukesh Kumar Raval*

Department of Chemistry, Gangadhar Meher College, Sambalpur, 768004, Odisha, India

*For correspondence

Prof. Mukesh Kumar Raval,

Retired Professor of Chemistry Gangadhar Meher College, Sambalpur, 768004 Odisha, India.

Email: mraval@yahoo.com

 

 

 

 

 

 

 

Received: 26 April 2016

Accepted: 14 May 2016

ABSTRACT

Objective: Hexokinases in the glycolytic pathway exhibit enhanced catalytic activity in malignant cells. In order to control cancer cell growth, the activity of the enzyme needs to be dampened. In the present study, an in silico approach is made to find potential phytochemical inhibitors of hexokinase I and II.

Methods: ArgusLab software is used to study the binding affinity of ligands in the active site of the protein. Computation of drug-likeness and oral toxicity is done using the online tools Molsoft and ProTox respectively.

Results: A flavonoid, 1, 2-dihydrobis (de-O-methyl)-curcumin, shows better binding affinity to the substrate as well as cofactor binding sites of both hexokinase I and II. The LD50 value (median lethal dose) is 2000 and the toxicity class is 4. There is no tox related fragments found in this structure and no possible binding to any toxicity targets.

Conclusions: 1, 2-dihydrobis (de-O-methyl)-curcumin is the inhibitor of choice, a lead molecule against targets: hexokinase I and II. The lead molecule may be the future drug for cancer treatment.

Keywords: Hexokinase-I and II, Cancer, 1, 2-dihydrobis (de-O-methyl)-curcumin, Drug-likeness, Toxicity, Molsoft, ProTox

Introduction

Cancer is the most dreaded diseases of the era. According to estimates from the International Agency for Research on Cancer (IARC) it strikes more than one-third of the world's population and cause more than 20% of all deaths.

Cancer is associated with abnormal cell division and growth. Normal cell division and growth depends upon the glycolytic pathway for energy. However, activity of enzymes in glycolysis is abnormally enhanced in cancerous cells. It fuels the cells to divide and grow at an abnormal rate. Curbing the glycolytic activity may reduce the growth of cancerous cells. Therefore, enzymes in the glycolysis pathway are studied as targets to develop novel cancer therapeutic agents.

The glycolytic hyper activity can be caused by over expression of hexokinases, primarily hexokinase-I and hexokinase-II1. Several studies demonstrate that hexokinase, particularly the type II isoform (hexokinase-II), plays a critical role in initiating and maintaining the high glucose catabolic rates of rapidly growing tumors. Most immortalized and malignant cells display increased expression of hexokinase-II, which might contribute to elevated glycolysis.2-4 Inhibition of this enzyme (hexokinase-II) is likely to have profound effect on cellular energy metabolism and survival. Thus, hexokinase is an attractive target for anticancer agents.

According to a report of World Health Organization, more than 80% of world's populations depend on traditional medicine for their primary health care needs.5,6 Plants have a long history of use in the treatment of cancer and it is significant that over 60% of currently used anti-cancer agents have come from natural sources.7 Several reports describe that the anticancer activity of medicinal plants is due to the presence of antioxidants in them. In fact, the medicinal plants are easily available, cheaper and possess no toxicity as compared to the modern synthetic drugs.8

The present work is basically the search for a potential phytochemical entity which can be useful in modulating the activity of hexokinase-I and II so that the rapid growth of cancer cells can be checked.

Materials and Methods

The target protein structure

The three dimensional coordinate files of target proteins hexokinase-I (1hkb.pdb) and hexokinase-II (2nzt.pdb) are obtained from the protein data bank (http://www.rcsb.org/pdb/).

The phytochemical compound database

The chemical structures of phytochemicals are obtained from Naturally Occurring Plant-based Anti-cancer Compound Activity Target database (NPACT) (http://crdd.osdd.net/raghava/npact/) are used as ligand library for search of hits.9

Preparation ligands for docking

Molegro Virtual Docker (MVD) is used for making the batch files of the ligands.

Docking ligands into the target protein active site

The protein-ligand interaction study is done using the software ArgusLab, which is a free software. Docking is performed in GADock engine using genetic algorithm with population size=50 and maximum generation= 1000. It predicts the ligand proximity with the active site of the protein on the basis of most negative binding energy called "Best Ligand Pose Energy".

Hexokinase isoforms have two domains each having two binding sites, one for glucose and other for ATP. So docking of ligands is done in glucose binding site as well as ATP binding site. The protein-ligand docking is performed according to the strategy given below:

Docking strategy

Situation-1: Docking of phytochemicals in the glucose binding site while the ATP binding site is preoccupied by ATP.

Situation-2: Docking of phytochemicals in the glucose binding site while the ATP binding site is unoccupied.

Situation-3: Docking of phytochemicals in the ATP binding site while the glucose binding site is unoccupied.

Situation-4: Docking of phytochemicals in the ATP binding site while the glucose binding site is preoccupied by glucose.

Top 5 molecules having best binding affinity with the active sites of hexokinase-I and II are selected as hits based on the scores (in kcal/mole) in all situations to both binding sites.

Prediction of drug- likeness

Prediction of molecular properties and drug-likeness is carried out with Molsoft (http://molsoft.com/mprop/). The online software calculates the molecular weight, number of hydrogen bond accepter (HBA), number of hydrogen bond donor (HBD), water partition coefficient (MolLogP), water solubility (MolLogS), Molecular Polar Surface Area (MolPSA), Molecular Polar Surface Volume (MolVol), number of stereo centers of the ligand and finally gives a value called "Molsoft Score". A ligand can be identified as a potential drug on the basis the molsoft score.

Toxicology study

The toxicity of the hits is predicted by the online software ProTox- Prediction of Rodent Oral Toxicity (http://tox.charite.de/tox/index.php? site=home). The toxicology study of input ligands are performed on the basis of toxicity prediction, toxicity classes, toxic fragments, toxicity targets, performance analysis.10,11

Designing of derivatives

The derivatives of the lead compound are designed and energy minimized in Hyperchem Pro 8.0, a molecular modelling software. The 3D structure of the derivatives are analysed by docking and there after prediction of drug likeness and toxicity.

Results and Discussion

The top five scoring ligands in each situation are taken into consideration for further analysis.

The 17 hits are then again docked in 1HKB and 2NZT in the same manner but this time high generation value of 10000. 1, 2-dihydrobis (de-O-methyl)-curcumin commonly showed the best binding affinity with hexokinase-I and hexokinase-II. Those are listed below in Table 1 and 2.

The bound form of 1, 2-dihydrobis (de-O-methyl)-curcumin in 1HKB and 2NZT is shown on the Figure 1 and Figure 2 respectively.

Table 1: Arguslab docking result of hexokinase-I.

HEXOKINASE-I (1HKB)
 

1HKB_Situation-1

1HKB_Situation-2

 

 

Molecule

Score

Molecule

Score

1

Altaicalarins A

-9.46

Ferruginin A

-9.99

2

1, 2-dihydrobis (de-O-methyl)-curcumin

-9.06

Garcinone E

-9.93

3

1,2-Dihydro-1,2,3-trihydroxy-9-(4-methoxyphenyl)phenalene

-8.33

1, 2-dihydrobis (de-O-methyl)-curcumin

-9.64

4

6-(2-Hydroxy-3-methyl-3-butenyl)-8-prenyl-eriodictyol

-8.09

Panduratin A

-8.94

5

Balanophonin

-7.42

Balanophonin

-8.91

 

1HKB_Situation-3

1HKB_Situation-4

 

Molecule

Score

Molecule

Score

1

Kaempferol-7-O-beta-d-glucoside

-11.24

3,5,7,3',4'-pentahydroxyflavonol-3-O-beta-D-glucopyranoside

-9.84

2

Isochamuvaretin

-10.66

Isochamuvaretin

-9.84

3

1, 2-dihydrobis (de-O-methyl)-curcumin

-10.19

Gericudranin C

-9.83

4

Malvidin-3-glucoside

-9.98

1, 2-dihydrobis (de-O-methyl)-curcumin

-9.48

5

Dysosmarol

-9.81

Kaempferol-7-O-beta-d-glucoside

-9.32

Table 2: Arguslab docking result of hexokinase-II.

HEXOKINASE-II (2NZT)
 

2NZT_Situation-1

2NZT_Situation-2

 

Molecule

Score

Molecule

Score

1

1,2-Dihydro-1,2,3-trihydroxy-9-(4-methoxyphenyl)phenalene

-9.74

Garcidepsidone A

-8.26

2

Lupinifolin

-9.35

1, 2-dihydrobis (de-O-methyl)-curcumin

-8.2

3

Garcinone D

-9.23

3. Gericudranin C

-8.18

4

1, 2-dihydrobis (de-O-methyl)-curcumin

-9.04

1-[3-(4-hydroxyphenyl)-2-propenoate]-beta-D-glucopyranoside

-8

5

Dysosmarol

-8.7

1,2-Dihydro-1,2,3-trihydroxy-9-(4-methoxyphenyl)phenalene

-7.98

 

2NZT_Situation-3

 

2NZT_Situation-4

 
 

Molecule

Score

Molecule

Score

1

1-[3-(4-hydroxyphenyl)-2-propenoate]-beta-D-glucopyranoside

-10.74

Kaempferol-5-O-beta-D-glucopyranoside

-9.96

2

1, 2-dihydrobis (de-O-methyl)-curcumin

-10.09

Kaempferol-3-O-beta-D-glucopyranoside

-9.92

3

3,5,7,3',4'-pentahydroxyflavonol-3-O-beta-D-glucopyranoside

-9.71

1, 2-dihydrobis (de-O-methyl)-curcumin

-9.9

4

Garcinone E

-9.32

4. 3,5,7,3',4'-pentahydroxyflavonol-3-O-beta-D-glucopyranoside

-9.77

5

Amygdalin

-9.2

Amygdalin

-9.37

Figure 1: 1, 2-dihydrobis (de-O-methyl)-curcumin docked into glucose binding site of 1hkb.

Figure 2: 1, 2-dihydrobis (de-O-methyl)-curcumin docked into glucose binding site of 2nzt.

Table 3: Interaction of 1, 2-dihydrobis (de-O-methyl)-curcumin in 2NZT.

Interaction of 1, 2-dihydrobis (de-O-methyl)-curcumin in Situation-1 of 2NZT
Ligand Receptor Residue Type

Distance(Å)

H 13411 OD 9546 ASP 657 H-donor

1.21

H 13412 O 13116 ASP 895 H-donor

1.61

O 13389 O6 13386 GLC 1003 H-donor

1.75

O 13388 OG 7854 THR536 H-acceptor

2.60

O 13388 N 7846 THR536 H-acceptor

3.18

O 13390 N 7860 ASN537 H-acceptor

2.91

O 13389 NZ 9062 LYS621 H-acceptor

2.99

O 13389 O6 13386 GLC 1003 H-acceptor

1.75

Interaction of 1, 2-dihydrobis (de-O-methyl)-curcumin in Situation-4 of 2NZT
Ligand Receptor Residue Type

Distance(Å)

O13366 OG7854 THR536 H-donor

2.58

O13368 OG9038 THR620 H-donor

2.97

O13367 O9867 GLY681 H-donor

2.61

O13368 OE10256 GLU708 H-donor

2.43

O13366 OG7854 THR536 H-acceptor

2.58

O13368 OG9038 THR620 H-acceptor

2.97

O13367 N9861 GLY681 H-acceptor

2.61

O13368 OE10256 GLU708 H-acceptor

2.43

Figure 3: Depiction of the interaction of 1, 2-dihydrobis (de-O-methyl)-curcumin with the active site residues of 2NZT in Situation-1.

1, 2-dihydrobis (de-O-methyl)-curcumin is taken as lead as it is showing good and common binding affinity in 1HKB and 2NZT binding sites with molsoft drug-likeness model score of -0.71 which is within the drug like range.

Figure 4: Depiction of the interaction of 1, 2-dihydrobis (de-O-methyl)-curcumin with the active site residues of 2NZT in Situation-4.

The toxicity of 1, 2-dihydrobis (de-O-methyl)-curcumin is then tested using an online tool called ProTox. The result is as follows:

Predicted LD50: 2000 mg/kg

Predicted Toxicity Class: 4

Average similarity: 57.39%

Prediction accuracy: 67.38%

Toxic fragments: No tox related fragments in input structure found.

Toxicity targets: No possible binding to toxicity targets.

Figure 5: Molsoft drug-likeness graph with score for 1, 2-dihydrobis (de-O-methyl)-curcumin.

Figure 6: 2D structure of 1, 2-dihydrobis (de-O-methyl)-curcumin.

The derivatives of 1, 2-dihydrobis (de-O-methyl)-curcumin are then designed by build module of Hyperchem software by structural alteration. The derivatives are listed below.

Derivative-1: (C19H18O4) - (1E)-1,7-bis(4-hydroxyphenyl)hept-1-ene-3,5-dione.

Derivative-2: (C25H30O9) - (6Z)-1,7-bis(4-hydroxyphenyl)-5-{[3,4,5-trihydroxy-6-(hydroxyl methyl) oxan-2-yl]oxy}hept-6-en-3-one

Derivative-3: (C25H30O9) - (6E)-5-hydroxy-7-(4-hydroxyphenyl)-1-(4-{[3,4,5-trihydroxy-6- (hydroxymethyl)oxan-2-yl]oxy}phenyl)hept-6-en-3-one

Derivative-4: (C31H40O14) - (6E)-5-hydroxy-1,7-bis(4-{[3,4,5-trihydroxy-6-(hydroxylmethyl)

oxan-2- yl]oxy}phenyl)hept-6-en-3-one

Derivative-5: (C19H17NO3) - 4-(2-{5-[(E)-2-(4-hydroxyphenyl)ethenyl]-1,2-oxazol-3-yl}ethyl) phenol

Derivative-6: (C19H18N2O2) - 4-(2-{5-[(E)-2-(4-hydroxyphenyl)ethenyl]-1H-pyrazol-3-yl}ethyl)phenol

The 2D structures of the 1, 2-dihydrobis (de-O-methyl)-curcumin derivatives are as follows in Figure 7.

(i) Derivative-1.

(ii) Derivative-2.

(iii) Derivative-3.

(iv) Derivative-4.

(v) Derivative-5.

(vi) Derivative-6.

Figure 7 (i – vi): Derivatives of the lead compound 1, 2-dihydrobis (de-O-methyl)-curcumin.

The energy minimized structures of 1, 2-dihydrobis (de-O-methyl)-curcumin Derivative are then docked in the binding sites of 1HKB and 2NZT in the same manner. The result is presented in the Table 4.

Table 4: Docking result of 1, 2-dihydrobis (de-O-methyl)-curcumin derivatives.

 

1HKB Situation

2NZT Situation

 

1

2

3

4

1

2

3

4

1,2-dihydrobis (de-O-methyl)-curcumin

(Lead)

-9.06

-9.53

-10.19

-9.48

-9.04

-8.20

-10.09

-9.90

Derivative-1

-8.09

-7.6

-7.77

-8.76

-9.32

-8.5

-7.42

-8.74

Derivative-2

-3.28

-4.19

-10.61

-9.93

-8.4

-4.26

-8.23

-10.22

Derivative-3

-6.69

-9.81

-8.41

-7.8

-8.09

-7.9

-8.16

-9.66

Derivative-4

2.99

-1.25

-6.36

-9.05

-8.3

-2.19

-7.25

-5.02

Derivative-5

-7.81

-6.88

-8.34

-7.56

-8.35

-9.19

-6.69

-7.52

Derivative-6

-8.15

-8.12

-7.56

-7.25

-9.06

-9.38

-7.46

-8.49

Table 5: Molsoft scores and ProTox Results of 1, 2-dihydrobis (de-O-methyl)-curcumin derivatives.

1,2-dihydrobis (de-O-methyl)-curcumin Derivatives

Molsoft Score

LD50 (mg/kg)

Toxicity Class (1-6)

Toxicity Target

Derivative-1

-1.04

2830

5

No tox related fragments

Derivative-2

-0.45

5000

5

No tox related fragments

Derivative-3

-0.78

3750

5

No tox related fragments

Derivative-4

-0.95

3750

5

No tox related fragments

Derivative-5

-0.17

1000

4

No tox related fragments

Derivative-6

-0.18

616

4

No tox related fragments

1,2-dihydrobis (de-O-methyl)-curcumin is a flavonoid extracted from Curcuma longa12 and the seeds of Alpinia blepharocalyx.12,13 Its antiproliferative activity is tested in cancer cell lines colon 26-L5 carcinoma and human HT-1080 fibrosarcoma cells which shows a remarkable anti-cancerous activity.12 As 1,2-dihydrobis(de-O-methyl)-curcumin and its derivatives have potential anti-cancer activity, they may further be studied in vitro and in vivo.

The results suggest that 1, 2-dihydrobis (de-O-methyl)-curcumin is a lead compound for anti-cancer drug development. The single molecule acts as inhibitor to both hexokinase I and II. The Molsoft drug likeness score is somewhat on the lower side, but prediction of toxicity by ProTox shows low toxicity (class 4, LD50 2000 mg/kg body weight). Therefore, 1, 2-dihydrobis (de-O-methyl)-curcumin can be further studied for development of ant-cancer treatment.

The derivatives of 1, 2-dihydrobis (de-O-methyl)-curcumin are also showing better binding affinity as compared to the lead in different situations of hexokinase I and II. Derivative-1 is scoring better than the lead in situation-2 of hexokinase II while derivative-2 in situation-3 and 4 of hexokinase I and situation-4 of hexokinase II. Derivative -3 and 5 are showing better result than the lead in situation-2 of both hexokinase I and II. Derivative-5 and 6 are scoring well in situation 1 and 2 of hexokinase II. This information implies that 1, 2-dihydrobis (de-O-methyl)-curcumin as well as its derivatives can be further analysed for developing a better drug for the modulation of the activity of hexokinases. The ProtTox analysis further says that the derivatives and the lead itself are orally safe as a drug as they have no fragments which are toxic in any manner and the higher LD50 values of the derivatives infer about the wide range doses as future drugs.

In case of malignancy, excessive expression of hexokinase, particularly the type II isoform, catabolises the glucose conversion to glucose-6-phosphate at high rates.14-17 This enhanced metabolism not only increases the production of biosynthetic precursors essential for cell growth, but maintains a high rate of ATP production under low oxygen (hypoxic conditions). 1, 2-dihydrobis (de-O-methyl)-curcumin is the phytochemicals which shows optimal binding affinity in both hexokinase-I and II. Thus it could be a potential drug which can modulate the activity of hexokinase-I and II.

Funding: No funding sources

Conflict of interest: None declared

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