INTRODUCTION:

Carcinomais known as the frequently occurringdisease worldwide caused by genetic mutations and damage by various intrinsic and extrinsic factors. This causative element alters the sequence and they accumulate together to stimulate cancers progression. Chronic lymphocytic leukemia (CLL) occurs due to abnormal behaviour of mature non-functional B lymphocytes. These cells excessively accumulate and proliferate in the bone marrow and lymph nodes^1,2.

CLL progression results in malignancies³ and other complications such as pancytopenia, infections, haemorrhage⁴ thrombocytopenia and low immunoglobulin concentrations⁵. CLLis mostly diagnosed in adults and elderly⁶. Prognostic markers help in the investigation of aggressiveness of the disease. In CLL the mutagenicity of the immunoglobulin heavy-chain variable-region (I_gV_H) confines with lower survival rates for CLL patients with unmutated I_gV_H⁷. The unmutated I_gV_H B cells show an increased expression of ZAP-70 (zeta-chain-associated protein 70) and are called as ZAP-70⁺B cells⁸.

The ZAP-70⁺B cells showed increased survival, poor prognosis and proliferative factors due to enhanced B cell receptor (BCR), CD8, CCR7 signalling⁹.The B cell receptor (BCR) signalling and their part in coordinating the crucial biological behaviour have elucidated on candidate protein kinases that are apparently stimulated in CLL^10,11. BCR pathway activation leads to the up regulation of the proliferative, anti-apoptotic proteins which are required for the progression of the disease.ZAP-70 refers to a protein kinase, containing three domains, two SH2 domains and one kinase domain (C-terminal). ZAP-70 is normally found in the T cells and absent in the B cells, but is expressed in CLL B cells¹².Hence targeted remedy for inhibiting the expression of ZAP-70 is an effective treatment to inactivate BCR pathway resulting in apoptosis of CLL cells. Some groups of drugs are used to terminate the cancer cells and someconstrain the advance of cancer cells^13,14,15.

Many of the drugs have multiple side effects and mild to severe reactions. Hence new anticancer agents should be investigated from various natural resources. There are several references from the marine resources showing the isolation of cytotoxic antitumor and there are several promising antitumor agents that have been isolated from marine macroalgae or seaweeds¹⁶. The biological activities displayed by macroalgae are immunosuppressant, antitumor, antimicrobial and antiviral activities which showed noticeable therapeutic effects in the biomedical investigations. Many anticancer compounds are obtained from macroalgae^17,18.Seaweed compounds is advantageous because of availability, low toxicity, which is suitable for oral applications along with a great variety of mechanisms of actions^19,20,21.

In present research ZAP-70 is targeted and found a seaweed compound which binds strongly to ZAP-70 active site though insilico docking studies. Molecular docking helps to explore the binding of the ligand or inhibitor and its interactions²². This information is generated to develop new drugs to inhibit the target protein. ZAP-70 protein can initiate apoptosis since the signalling for cell survival is inhibited. Such natural compounds are efficient and can be potential ZAP-70 inhibitors and used for CLL treatment²³.

MATERIALS AND METHODS:

Protein Selection and Preparationl:

The crystallographic co-ordinates file for wild-typeZAP-70 protein was obtained from Protein Data Bank (PDB ID: 2OZO). Protein establishment was done with the aid of protein preparation wizard^24,25,26. Protein was then optimized; H₂O fragments were eliminated and it was minimized using OPLS (2005) force field²⁷.

Active Site Prediction:

The active site (binding pocket) of ZAP-70 was analysedand outlined using SiteMap module by Schrodinger Maestro suite. Sitemap calculation was performed with the initial search that analyses using grid points with multipledomains on the proteins surface which is suitable for binding ligands to the receptor²⁸.

Ligand Preparation:

Seaweed metabolite database (SWMD) (http://www.swmd.co.in) is a database which provides information of compounds from seaweeds with descriptive fields²⁹. Each access in the database is categorized into sections such as general information, structure information, predicted properties and bibliographic references and unique SWMD accession number. Ligands were enhanced with the aid of LigPrep module of the Schrodinger Maestro suite^29,30,31.

High-Throughput Virtual Screening and Molecular Docking Analysis:

High-throughput virtual screening of compounds was observed in contrast to ZAP-70 using a GLIDE moduleof Schrodinger Maestro suite^31,32. The active site was preferredand selected from sitemap predicted score. The best poses for the ligand with the protein were finalized based on the Glide Score. The ligand with highest binding score was confirmed to be the optimal ligand. In the current screening process, sequential decline of ligand hits was reporteddepending on implication of glide score³².

Molecular Dynamics Analysis:

The stability of the docked complex was estimated through molecular dynamic simulations using GROMACS version 4.6.5 with Gromos43a1 force field²³. The input for running simulation is by providing the structure coordinate file for the protein. The protein system is solvated by the addition of SPC216 (Simple point charge) H₂O entities. The recurring edge condition was added by generating a box around the protein system²⁴. The system was neutralized by the addition of Sodium or Chloride atoms using the genion tool. The structure was energy minimized by steepest descent method for 50000 steps. Once the protein is minimized, the system was equilibrated at constant temperature of 300K and constant pressure of 1 bar with a LINCS constraint for all bonds²⁶. Berendsen thermostat coupling method with a view to keep the temperature constant inside of the simulation box²⁴. Particle Mesh Ewald method investigated the electrostatic interactions²⁷.Finally, the production MD was performed for the protein ligand complex and apo protein of ZAP-70.Stability and structural analysis of MD simulation was performed using GROMACS utilities and the trajectories were obtained for the protein-ligand complex and Zap-70 protein. The Root Mean Square Deviation (RMSD) and the Inter Hydrogen bond (NH) formed between ligand and protein were obtained through g_rmsd and h_hbond.

RESULT AND DISCUSSION:

Protein Selection and Preparation and Effective Site Investigation

The ZAP-70 crystal structure (PDB Id: 2OZO) was utilised for the research work and prepared with the aid of Protein Preparation Wizard which removes the water molecules, added bond orders using OPLS force field. The protein was optimized through protein minimization to achieve minimal energy conferred to the stable protein. Any hetero atoms present were removed. The reduced arrangement was used in Sitemap to find the effective pockets which has highest volume and the pocket consisting of the protein kinas activity in ZAP-70 was selected. This pocket grid was used for docking of the legend entities. The leukemia is the abnormal growth of the white blood corpuscles and CLL is a tumor of the B cells when there is auto reaction in the mature B cells³⁵. The B cell receptor pathway is activated and up regulates all the proteins required for the survival of the cells³⁶. This occurs due to the over expression of the tyrosine kinase protein ZAP-70 and so this protein is used as the target. The protein activates the NK-cells and the T-cells and is not present in the peripheral B cells^36,37,38. Proteins which are unique and expressed exclusively in the disease condition is a target of interest and is ineffective on normal cells. Hence targeted therapies will help cure CLL³⁹.

Ligand Selection and Retrieval:

Seaweed Metabolite Database (SWMD) database consists compounds from marine macroalgae collected from literature. The database also contains all the information related to the compound properties and substances are present in downloadable form⁴⁰. The database initially contains 500+ entries and through the chemical descriptors calculation many conformers were accessible for each of the compound. A total of 1055 compound structures were available. These ligand 3D structures were obtained and were progressed utilising LigPrep module. (Schrödinger, LLC, New York, NY, 2015). The compounds excluded for high throughput virtual screening did not follow the Lipinski’s rule of five^41,42.

High Throughput Virtual Screening Using GLIDE:

Virtual screening enables to screen the molecules which bind efficiently to the protein 3D structure. Docking is the process by which the molecules fits into the pocket of the target protein and these insilico studies is essential. The screening and the filtering of the best fit compounds to the target protein is performed using HTVS (high throughput virtual screening) in GLIDE module of the Schrodinger Maestro suite (Schrödinger, LLC, 2015, New York, NY software). The effective site predicted using Sitemap module in the kinase activity domain. The goal of ligand-protein docking is done to investigate dominating binding model of a ligand with a protein of familiar3D structure. Depending on the glide score the docking result is analysed¹⁵.The virtual screening was performed in a series of High throughput virtual screening (HTVS) succeeded by Standard precision docking (SP) and finally Extra precision (XP) docking which filtered out the compounds binding weakly to ZAP-70 and the docked compounds which showed high binding scores are tabulated in Table 1Further, the 2D images generated were analysed for intermolecular interactions like H-bond formation³³.The molecular docking result is revealed that Methyl N, N’-bis-(2,3-dibromo-4,5-dihydroxybenz -yl)-y-ureidobutryate (RR017) has the best dock score of -10.279 kcal/mol. The 2D imagining of the docked complex revealed in Fig 1 shows that it contains 7 H-bonds within the protein and it is forming H-bonds with several amino acids like Gly418, Ala417, Asp479, Lys369 and Leu344.The hydrogen bond formed between the Lys369 and the H atom of the compound RR017. The Lys 369 residue is essential for the catalytic activity of ZAP-70.

Table 1: The compounds with highest glide score:

S. No	Compounds Name	Glide Score
1	RR017	-10.279
2	BE007	-8.752
3	RR005	-8.437
4	RR021	-8.327
5	RR021	-8.327

Figure 1: 2D plot of the compound RR017 interacting with pocket region of ZAP-70

Fig 2a) RMSD Plotwhere red line is complex ZAP-70 with RR017 and black line shows the unbound ZAP-70 protein b) Inter Hbond between protein and ligand

From the Molecular dynamics simulation studies for 10ns was performed to evaluate the stability of the docked protein complexin comparison to the unbound ZAP-70. From the Root Mean Square Deviation (RMSD) Plot the complex shows stability and converges at 0.4nm and deviated in a range of 0.2 – 0.45 nm. The inter Hydrogen bond plot to investigate the number of H-bonds formation between ligand and protein was obtained from molecular dynamic simulation studies showed that mean of 2-4 hydrogen bonds were formed for half of the simulation and mean of 1-2 were formed from 5 to 9ns. At 10ns a highest of 7 hydrogen bond were formed. Thus the compound RR017 which corresponds to the compound Methyl N, N’-bis-(2,3-dibromo-4,5- dihydroxybenz-yl)-y-ureidobutyrate gave the best binding to the kinase domain. An association between ZAP-70 and CLL cancer is suspected, since the it is over expressed in B cells. The current treatments for CLL (Chlorambucil, Fludarabine, anti-CD20 Rituximab, etc.) leads to cell death in CLL cells but resulting into immune-suppression and patients frequently generate drugresistance¹². Since no proper medication is available for CLL and to inhibit the cell survival and proliferation in these cells, the blocking of the signalling pathways through inhibition of Zap70 is a potential treatment for CLL⁴³ and the compound Methyl N,N’-bis-(2,3-dibromo-4,5-dihydroxybenz-yl)-y-ureidobutyrate can be used as a therapeutic agent for CLL.

CONCLUSION:

In this computational study, inhibitor search through molecular docking analysis for ZAP-70 was performed against the compounds obtained from Seaweed metabolite database using GLIDE module. The virtual screening process involved in GLIDE are High-throughput virtual screening, Standard precision docking and Extra Precision docking which filtered the compound bound strongly to the ZAP-70. The compound RR017 bound to the active site of ZAP-70 with a dock score of -10.279 kcal/mol. This dock score was the highest among the other compounds and the dock complex showed to be stable in comparison to the unbound ZAP-70 protein through molecular dynamics simulation studies. This compound is a potential lead compound for inhibiting the signalling involved in the cancer cell survival. Such targeted therapies help CLL management and improve survival rates. The Normal cells are not affected and using natural compounds for anticancer therapy can reduce the side effects of radiations and chemotherapy.

ACKNOWLEDGEMENTS:

The authors gratefully acknowledge VIT University, Vellore for the support through Seed Grant for Research.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 17.03.2017 Modified on 19.05.2017

Research J. Pharm. and Tech 2017; 10(12): 4162-4166.

DOI: 10.5958/0974-360X.2017.00758.2