In silico Molecular Docking studies to investigate interactions of natural Camptothecin molecule with diabetic enzymes

 

Jeyabaskar Suganya1*, Viswanathan T2, Mahendran Radha, Nishandhini Marimuthu1

1Department of Bioinformatics, School of Life Sciences, Vels University, Pallavaram, Chennai-17. TamilNadu, India

2 Department of Microbiology, LRG Govt. Arts College of Women, Tiruppur- 641 604, Tamil Nadu, India.

*Corresponding Author E-mail: suganyaj11@gmail.com

 

ABSTRACT:

The third leading cause of death in the world is Diabetes. Diabetes comes under a group of metabolic disorders in which the patients possess high blood glucose level. Camptothecin is an alkaloid compound with better antioxidant activity and in china it was used as traditional drugs to cure many diseases. The early researches on camptothecin revealed that it possess better anticancer and antitumor activity when compared with current available synthetic drugs. In silico drug designing and docking studies pave a way for better understanding of inhibitory activity of the compound over the respective target proteins. Molecular docking is one of the most powerful tools for predicting and analyzing the binding interactions between receptors and small molecules at the atomic level. In the current study, an in silico binding interaction of the compound camptothecin with three important diabetic targets like Glucokinase, insulin receptor and PPAR gamma were analyzed using Arguslab. ArgusLab software is used for molecular docking, to study the interactions between small compounds and its proteins. The compound camptothecin was first tested for its drug likeliness, bioactive properties and the results predicted that the compound passed the test for being an oral drug. The docking study revealed that camptothecin showed best binding affinity towards all the three proteins.  Among the three proteins, PPAR gamma protein exhibited the lowest binding energy, followed by Glucokinase and insulin receptor with the binding energy of > - 8.5 kcal/mol. Thus, Camptothecin compound could be considered as a better lead molecule in the development and discovery of new anti-diabetic drugs.

 

KEYWORDS: Camptothecin, Diabetes, Glucokinase, Insulin receptor and PPAR gamma, docking.

 

 

 


INTRODUCTION:

Diabetes mellitus is a chronic metabolic disorder with altered carbohydrate, lipid and protein metabolism1. In 2011, about 366 million people suffered with diabetes globally and is expected to increase up to 552 million by 20302. One recent study by ICMR-INDIA reported that about 62.4 million type-II diabetic people are from India and expected to increase up to 101 million by the year 20303. A change in diet, lifestyle and exercise will help to a great extent in management of diabetes at the early stages with little lesser impact at the later stages of life.

 

Several drugs such as biguanides and sulfonylureas are presently available to reduce hyperglycemia in patients with Diabetes mellitus. These drugs have side effects like kidney, liver damage and thus searching for new biologically active compounds are essential to overcome Diabetes mellitus4. Plant-based products have been popular all over the world for centuries. In diabetic patients, some herbal alternatives are proven to provide symptomatic relief and assist in the prevention of the secondary complications of the disease. Some of the herbal preparations seem to regenerate beta cells also. Thus the need of basic information about the availability of anti-diabetic plants in India for researchers for their scientific quest is fanatically felt. The ethnobotanical reports state that about 800 plants from India may possess anti-diabetic potential5.

Camptothecin is an aquinoline alkaloid plant compound which was first isolated from the bark and stem of Camptotheca acuminata, a tree native to China used as a cancer treatment in Traditional Chinese Medicine6. Further analysis of Camptothecin compound revealed the presence of antioxidant, anti-inflammatory, immunomodulatory, antitumor and antiproliferative anti-diabetic activity7. On survey of the compounds, the various plants possess camptothecin and in the current study camptothecin was docked with diabetic proteins like glucokinase, peroxisome proliferator-activated receptor gamma (PPARγ), and insulin receptors using Bioinformatics tools8.

 

The treatment of diabetes involves in lowering the blood glucose level through various mechanisms, which includes insulin secretion, glucose absorption and glucose metabolism9. The three main protein involved in theses metabolism were glucokinase, PPARγ, and insulin receptors. Glucokinase is a cytoplasmic enzyme present in the liver and pancreas. Its main function is regulation of glucose levels in these organs10, 11. PPARγ protein is the nuclear receptor transcription factor and it also regulates target genes which involved in the process of glucose and lipid homeostasis12,13. Another important insulin receptor was the protein tyrosine phosphatase 1B which has effect over insulin sensitivity which results in modulating insulin signal transduction14, 15.

 

The drug molecule is triggered when the binding of small molecule to the receptor protein is perfectly done. Such protein-ligand interaction is comparable to the lock-and-key principle, in which the lock encodes the protein and the key is ensembled with the ligand. The major driving force for binding appears to be hydrophobic interaction whose specificity is however controlled by hydrogen bonding interactions16.

 

MATERIALS AND METHODS:

Preparation and evaluation of diabetic inhibitor:

The Camptothecin compound was identified and retrieved by using the Pubchem compound database and saved in.sdf format17. By using the Molinspiration server, the drug likeness and bioactive properties of the camptothecin compound was analyzed on the basis of “Lipinski's Rule of Five”18.  The rule states that if the compound could be used as an  oral active drug then the compound should satisfy the following criteria: a) molecular weight : below 500 Daltons, b) Log P: less than 5, c) Hydrogen bond donors: less than 5 and d)hydrogen bond acceptors: less than 1019. The bioactive properties predict 6 bioactive properties such as follows: GPCR ligand, Ion channel modulator, Nuclear receptor ligand, Protease inhibitor, Enzyme inhibitor and Kinase inhibitor20.    All the bioactive properties were predicted by its score. If the score lies between -0.50 to +0.50, then the compounds can consider for oral drugs.

 

Preparation of Diabetic Protein:

The three dimensional crystal structures of the target Glucokinase, insulin receptor and PPAR gamma along with  its sequence in fasta format and PDB IDs: 1V4T, 1IR3, 3DZY were retrieved from the protein data bank (PDB) (http://www.rcsb.org/pdb/) which was determined by X-Ray crystallography. 21-23

 

Determination of active site and secondary structure of diabetic proteins:

The identification of catalytic sites present in the Glucokinase, insulin receptor and PPAR gamma was carried out with Computed Atlas of Surface Topography of Proteins (Castp) program24,25.The prediction of secondary structures present in the targets was performed by submitting its fasta sequence to CFSSP(Chou and Fasman Secondary Structure Prediction) database26,27.

 

Visualization of diabetic protein and its inhibitor:

The three dimensional structure of target proteins were viewed using the molecular visualization tool PyMoL28. The minimized 3D structures were saved in PDB file format with presence of hydrogen bonds in all polar residues and its fasta sequence. The two dimensional structure of the compounds were viewed in SDF file, then its energy form were minimized and converted to pdb format.  Now both the protein and ligand were ready for docking analysis.

 

Docking Studies Using ArgusLab:

Docking analysis between the proteins and ligand was performed by Arguslab docking software which is the most commonly available software30. For docking purpose, all parameters in Arguslab were selected by default. . Docking analysis was performed using “Argus Dock” with grid resolution-0.40Å, docking precision-“Regular precision” and “Flexible ligand” were set for docking mode was employed for each docking run31. The docking stability of the ligand and protein was evaluated using Argus Lab energy calculations (Kcal/mol) with the number of hydrogen bonds formed between them32.

 

RESULTS AND DISCUSSION:

Preparation and evaluation of diabetic inhibitor:

By analyzing the results of Molinspiration, it was identified that the compound has passed through the Lipinski’s rule of five criteria by satisfying all bioactive properties are shown in the Table 1 and 2 and this result confirmed that the compound can be used as an oral drug. The two and three dimensional structure of camptothecin was retrieved with its Pubchem ID and molecular formula (Table 3).


 

Table 1: The compound Camptothecin satisfies the Lipinski's Rule of Five

Smiles

Molecular Weight

Partition Co-Efficient Log P

Hydrogen Bond Donors

Hydrogen Bond Acceptors

CCC1(C2=C(COC1=O)C(=O)N3CC4=CC5=CC=CC=C5N=C4C3=C2)O

348.36

1.434

1

6

 

Table 2: The compound Camptothecin satisfies the bioactive properties.

Bioactivity

Properties

GPCR ligand

Ion channel modulator

Kinase inhibitor

Nuclear receptor ligand

Protease inhibitor

Enzyme inhibitor

Scores

0.16

-0.15

0.08

0.07

-0.10

0.11

 

Table 3: The two and three dimensional structure of the compound Camptothecin.

2D structure of compound

3D structure of compound

Pubchem ID:     24360

 

 

Molecular  Formula:  C20H16N2O4

 

 

Table 4: The secondary structure of target proteins using CFSSP.

1V4T: Sequence length – 455

1IR3 : Sequence length – 451

3DZY :  Sequence length – 467

Secondary structure     Amino Acid      %

Secondary structure     Amino Acid      %

Secondary structure      Amino Acid       %

Alpha helix (Hh)  

Extended strand  (Ee)      

Beta turn (Tt)     Random coil   (Cc)

216

82

51

106

47.47

18.02

11.21

23.30

Alpha helix (Hh)      Extended strand (Ee)

Beta turn (Tt)               Random coil    (Cc)

 212 

 82

51

106

47.01

18.18

11.31

23.50

Alpha helix     (Hh)

Extended strand (Ee)

Beta turn       (Tt)    Random coil     (Cc)

163

67

37

200

34.90

14.35

7.92

42.83

 

 

 

 

 


Analysis of the active sites and secondary structure of the targets of different types of diabetes:

The secondary structure and catalytic site predictions for diabetic targets was analyzed using CFSSP and Castp program. The percentages of secondary structure present in target proteins (Table 4) and the best catalytic sites for ligand binding were observed in the target proteins (Table 5)

 

Docking Analysis using Argus Lab:

Docking studies were carried out using predicted active residues of the target proteins with Camptothecin. The results of the interaction with protein and its binding energy formed during hydrogen interaction to compound Camptothecin were saved in PDB file format. The docking results were further analyzed using pymol and following result were predicted.

 

Camptothecin with PPAR gamma protein:                                                                                                  

Among the proteins, PPAR gamma protein exhibited the least binding interaction of –9.83 Kcal/mol with Camptothecin. Camptothecin was bound to the active site of ARG 159 with hydrogen bond length of 2.54Å Figure 1. The hydrogen bond was formed between the atoms of NH1 present in the PPAR gamma protein to atom of OP1 in Camptothecin36. Thus, the compound has the capacity to activate the process of glucose and lipid homeostasis in diabetic conditions.


 

 

Table 5: The active sites of the target proteins prsdicted using CASTP

Target

Active site  present in the structure

Active site  present in the sequences

1V4T

 

 

1IR3

 

 

3DZY

 

 

 


 

Fig1: Binding Energy of  Camptothecin with 3DZY –9.83 kcal/mol.

Indicates the active site of the PPAR gamma protein

Indicates the Camptothecin

Indicates the binding atom

Indicated the hydrogen diatance in Å

 

 

Fig. 2: Binding Energy of Camptothecin with 1V4T– 9.12 kcal/mol.

Indicates the active site of the glucokinase

Indicates the Camptothecin

Camptothecin with Glucokinase:

Glucokinase exhibited the second least binding energy of -9.12 kcal/mol but no hydrogen bond interaction was explored with the compound camptothecin even though the best interaction were occurred Figure 2. The result suggested that camptothecin has the capacity to monitor the glucose levels in diabetic patients36

 

Camptothecin with Insulin receptor:

The enzyme was bound to Camptothecin at the active site of Arg1131 with a docking energy of -8.67 kcal/mol Figure 3. During the docking process, two hydrogen bond was formed with the single oxygen atom of the compound with the atom of NH1 and NH2 of the active site of protein at the distance of 2.88 Å and 3.00 Å. The predicted interaction suggested that the compound possess ability to control insulin secretion.36

 

The in vitro studies on Camptothecin revealed that the compound plays a vital role as a potent inhibitor for cancer, tumor, inflammation, and also act as a good antioxidant33-35. From the insilico docking results, it is quite evident that plant compound Camptothecin also bears a potential anti-diabetic activity over major diabetic target enzyme.


 

Fig3: Binding Energy of Camptothecin with 1IR3– 8.67 kcal/mol.

Indicates the active site of the Insulin receptor

Indicates the Camptothecin

Indicates the binding atom

Indicated the hydrogen diatance in Å

 


CONCLUSION:

In silico docking studies revealed that the natural compound camptothecin served as the best natural potent inhibitor for diabetes and also satisfied the Lipinski rule of five and bioactive properties. The outcome of the present study could be useful for identifying, developing and designing novel preventive and therapeutic anti-diabetic drug with better pharmacological activity which possess the best inhibitory activity against potent diabetic proteins like PPAR gamma protein, glucokinase and insulin receptor. In silico docking showed that the camptothecin was capable of binding efficiently with various targets 3DZY, 1V4T, and 1IR3. Lower docking energies of camptothecin (with 3DZY –9.83 kcal/mol, 1V4T– 9.12 kcal/mol, 1IR3– 8.67 kcal/mol) with the active amino acid residues (ARG 159 – 3DZY, Arg1131- 1IR3) during the hydrogen interaction. Camptothecin bound to the functional site of the proteins, there by inhibiting the protein function directly which results in maintaining the blood glucose level in diabetic patients. The result of the current work provides strong recommendation for the compound camptothecin to be considered as a novel oral drug for diabetics. Further this study can be implemented for the development of valuable lead drug candidates for the treatment of diabetes and dosage of safety level of the compound camptothecin could be determined by clinical trials.

 

CONFLICT OF INTEREST:

The authors declare they have no competing interests.

 

ACKNOWLEDGEMENT:

We acknowledge Vels Institute of Science, Technology and Advanced Studies (VISTAS) for providing us with required infrastructure and support system needed.

 

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Received on 08.03.2017          Modified on 23.03.2017

Accepted on 15.04.2017        © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(9): 2917-2922.

DOI: 10.5958/0974-360X.2017.00515.7