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INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407

Research Article

COMPUTATIONAL EVALUATION OF SPIROISOXAZOLINES AS INHIBITOR OF AChe Gaikar Rajendra Babasaheb1, Vikhe Pratik Prakash1, Gadhave Anil Gorakshanath2,

Karale Bhausaheb Kisan3* 1Center for Biotechnology, Pravara Institute of Medical Sciences (DU), Loni, Dist. Ahmednagar, Maharashtra, India

2P.V.P. College Pravaranagar Loni, Dist. Ahmednagar Maharashtra, India 3Department of Chemistry, Radhabai Kale Mahila Mahavidyalaya, Ahmednagar 414001, Maharashtra, India

Article Received on: 10/04/12 Revised on: 25/05/12 Approved for publication: 03/06/12

*Email: bkkarale@yahoo.com ABSTRACT Spiroisoxazolines are structurally diverse group of compounds that show wide range of biological activity. The computational evaluation of the spiroisoxazolines derivatives as the acetylcholinesterase inhibitor was performed. By using online servers applying different approaches the acetylcholinesterase was identified as the target for the synthesized derivative. The docking analysis was performed by PyRx software using AutoDock 4. Further the molecular descriptor analysis was performed to identify drug likeness of the derivatives by Lipinski’s rule of five. Derivatives showed comparatively significant binding energy values and followed the Lipinski’s rule. The study provides the base for further in vitro and in vivo study of the spiroisoxazolines derivatives as acetylcholinesterase inhibitor and proposed drug to be used for Alzheimer disease. Keywords: Spiroisoxazolines, acetylcholinesterase(AChe), Molecular docking, Molecular descriptor, Lipinski’s rule of 5. INTRODUCTION Isoxazolines represent one of the most active classes of heterocyclic compound which possesses wide range of biological activities such as herbicidal, plant-growth

regulatory1, antitumor activity

2,3, antitubercular activity

4,5 as

well as antibacterial agents6. In the view of these reports the

synthesized spiroisoxazolines7 were subjected to insilico screening for identification of their probable biological target. Diverse approaches are used for the identification of potential target (protein) for the synthesized chemical derivatives. The proteomic approach which compares the protein expression profiles for a given cell or tissue in the presence or absence of the derivative and thus identifies the target. But the method has proved to be time consuming and arduous8. In current scenario the insilico drug target profiling is rising as an effective alternative for an high throughput and laborious invitro screening8,9. Also an activity referred to as a drug repurposing in which the old drugs are screened for a new targets is possible by this approach10,11. In current study we used three on line web servers which uses different computational approaches to identify the potential protein targets. The results suggested the acetylcholinesterase (AChE) as the potential target for the spiroisoxazolines derivatives screened. There is general interest in choline esterase inhibitors because they may be used in the treatment of Alzheimer’s disease12. Further the derivatives were subjected to molecular driscriptor analysis to determine drug likeliness and molecular docking analysis for the calculation of binding energies with the AChE. MATERIALS AND METHODS Ligand Structure Preparation The spiroisoxazolines derivatives earlier synthesized and the structural data identified by IR and NMR mass7 were used as ligand drug data set. ChemSketch13, the chemically intelligent drawing interface freeware (http://www.acdlabs.com/download) was used to draw the structures of spiroisoxazolines derivatives (Fig. 1), followed

by generation of structure in PDB and SDF format using “On Line SMILES Translator and Structure File Generator” (http://cactus.nci.nih.gov/services/translate/) utility which relies on CACTVS technology and utilizes the algorithm of program COoRdINAtes (CORINA)14,15 to generate 3D atomic coordinates of a molecule. Potential Target Identification Three online web servers were used to identify the potential targets (protein) for the spiroisoxazolines derivatives. Initial screening was done by predicting the bioactivity score at the molinspiration server provided at www.molinspiration.com. The server calculates the drug likeness score towards GPCR ligands, ion channel modulators, kinase inhibitors, nuclear receptor ligands, protease inhibitors and other enzyme targets using sophisticated Bayesian statistics method16. For further precise identification of the target protein the spiroisoxazoline derivatives files in SDF format were submitted to the Pharmmaper (http://59.78.96.61/pharmmapper/index.php) and ReverseScreen3D (http://www.modelling.leeds.ac.uk/ReverseScree n3D/index.html) servers. The Pharmmaper server uses the reverse phramacophore approach. The output of a PharmMapper run is demonstrated in the form of a ranked list of hit target pharmacophore models that are sorted by fit score in descending order17. The ReverseScreen3D server uses reverse virtual screening (VS) method called ReverseScreen3D. The method uses a 2D fingerprint-based method to select a ligand template from each unique binding site of each protein within a target database. The target database contains only the structurally determined bioactive conformations of known ligands. The 2D comparison is followed by a 3D structural comparison to the selected query ligand using a geometric matching method, in order to prioritize each target binding site in the database11. The output is in the form of a list of the 2D and 3D scores in descending order.

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Protein Molecule Preparation After the initial screening of the derivatives for the potential target identification the acetylcholinesterase was considered as best target and used for further analysis. The coordinate for the crystal structure of acetylcholinesterase was downloaded (PDB id- 1ACJ)18 form Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). The PDB file was prepared by only selecting the chain A of the protein molecule and saving the file in the absence of the ligand using the Swiss-PDB Viewer19. Also the ligand i.e. diphenolic azoles was selected and saved as separate PDB file to be used as reference ligand. The new acetylcholinesterase protein file (PDB), the reference ligand file (PDB) and the spiroisoxazolines derivatives PDB files were used for further molecular docking analysis. Docking Analysis Molecular Docking involves the rapid Computational assessment of most favorable interacting regions between two different molecules20. For docking study of spiroisoxazolines derivatives in acetylcholinesterase the PyRx virtual screening software for Computational Drug Discovery was used. It uses a large body of already established open source software such as Auto Dock 4 and Auto Dock Vina21 software’s for the docking study. Open Babel was used for energy minimization22. Initially, spiroisoxazolines derivatives were energy minimized using the steepest decent method23 with MMFF94 force field24 later subjected to molecular docking analysis using Autodock 4 by Lamarckain Genetic Algorithm. The binding energies were obtained for the spiroisoxazolines derivatives and the reference ligand. The binding pose (Figure 2) showing best score were visualized in PyMOL25. Calculation of Molecular Properties The molecular properties were calculated on basis of simple molecular descriptors used by “Lipinski’s rule of five"26. The five properties consist of Molecular weight, hydrogen donor; acceptors, LogP, and Total Polar Surface Area (TSPA) which were calculated using the online chemoinformatics tool molinspiration (http://www.molinspiration.com/). RESULT AND DISCUSSION In current study the potential target identification for spiroisoxazolines derivatives was done and the target was found to be acetylcholinesterase. Further evaluation of binding efficiency between acetylcholinesterase and spiroisoxazolines derivatives was done by using computational approach. The derivatives showed quality binding energy scores which illustrate that they have significant affinity towards acetylcholinesterase. Potential Target Identification To identify the potential target the spiroisoxazolines derivatives (SPIRO) were screened using three online servers and the scores were obtained as shown in Table 1. The Molinspiration bioactivity score was seen to be significantly high (above 0.10) except for SPIRO6 towards the enzyme inhibitor. The results obtained for the Pharmmaper server showed the high fit scores and rank above 150 for spiroisoxazolines derivatives as ligand of the acetylcholinesterase. Screening outcome of ReverseScreen3D also showed top rank and high scores (2D and 3D) for the acetylcholinesterase (PDB id: 1ACJ). Thus the acetylcholinesterase (PDB id: 1ACJ) was considered to be potential target for spiroisoxazolines derivatives and used for further molecular docking analysis.

Docking and Molecular Descriptor analysis Docking analysis was performed by PyRx virtual screening software using Autodock 4. The binding energies calculated are as shown in Table 2. The spiroisoxazolines derivatives 2 and 5 showed significant binding energy values compared to the reference ligand. The molecular descriptor study was performed on basis of “Lipinski’s rule of five" using the Molinspiration server. Lipinski’s Rule of Five is a rule of thumb to evaluate drug- likeness, or determine if a chemical compound with a certain pharmacological or biological activity has properties that would make it a likely orally active drug in humans. The rule was formulated by Christopher A Lipinski25. The rule describes molecular properties important for a drug’s pharmacokinetics in the human body, including their absorption, distribution, metabolism, and excretion (“ADME”). Rule states that, in general, an orally active drug has26, 27. ® Not more than 5 hydrogen bond donors (OH and NH

groups) ® Not more than 10 hydrogen bond acceptors (notably N

and O) ® Not more than 15 rotatable bonds (rotb) ® A molecular weight (M.W) under 500 g/mol ® A partition coefficient log P (mi.LogP) less than 5 All the spiroisoxazolines derivatives followed Lipinski’s rule. The molecular descriptor values obtained are as shown in the Table 3. Comparing the results it is evident that the spiroisoxazolines derivatives 2 and 5 show quality binding energy score and also follow the Lipinski’s Rule of Five to bring them closer to the drug like molecule. CONCLUSION Acetylcholinesterase (AChe) is the enzyme which is responsible for the breakdown of Acetylcholin in the neural synapse. The only group of drugs currently licensed for Alzheimer’s disease treatment is the AChe inhibitors28. In current study the target for the spiroisoxazolines derivatives was identified to be acetylcholinesterase and binding energy values were significant which showed the prediction to be precise. So indeed the study gives a platform for the further in-vitro and in-vivo analysis of the spiroisoxazolines as the inhibitor of the enzyme acetylcholinesterase. REFERENCES 1. Howe RK, Shelton BR. Spiroheterocycles from the reaction of nitrile

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Table 1: Potential Target Identification Scores for Spiroisoxazolines Derivatives

Moiety Molinspiration bioactivity score (Enzyme Inhibitor)

Pharmmaper Score (Acetycholinesterase)

Reverse 3D Score (Acetycholinesterase)

Fit Score Normalized Fit Score 2D score 3D score SPIRO1 0.10 3.49 0.4362 0.4962 0.5652 SPIRO2 0.12 3.29 0.4201 0.4888 0.5651

SPIRO3 0.13 3.42 0.4307 0.4583 0.5416

SPIRO4 0.13 3.10 0.4112 0.4583 0.5416

SPIRO5 0.19 3.86 0.4423 0.5000 0.5909

SPIRO6 0.06 3.01 0.4029 0.3122 0.2459

Table 2: Binding Energy and Molecular description score of for Spiroisoxazolines Derivatives

Moiety Binding energy (kcal/mol) LogP value Molecular Weight TPSA HA ON

HD OHNH Nrotb No. Of

Voilations SPIRO1 -8.24 4.782 300.7 34.49 3 0 1 0 SPIRO2 -9.80 4.782 300.7 34.49 3 0 1 0 SPIRO3 -8.75 4.390 316.7 43.72 4 0 2 0 SPIRO4 -8.26 4.390 316.7 43.72 4 0 2 0 SPIRO5 -9.38 4.357 286.7 34.49 3 0 1 0 SPIRO6 -7.40 4.729 343.4 39.42 4 0 3 0

Reference -8.00 --- --- --- -- --- -- ---

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Figure 1: Chemical structure of Spiroisoxazolines derivatives

N

ON

Cl

H3C

SPIRO1

N

ON

ClH3C

SPIRO2

SPIRO3SPIRO4

SPIRO5SPIRO6

N

ON

ClMeON

ON

Cl

MeO

N

ON

ClN

N

NO

Figure 2: Spiroisoxazolines derivatives docked in acetylcholinesterase

Spiroisoxazolines Derivative 1 Spiroisoxazolines Derivative 2

Spiroisoxazolines Derivative 3 Spiroisoxazolines Derivative 4

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Spiroisoxazolines Derivative 5 Spiroisoxazolines Derivative 6

Source of support: Nil, Conflict of interest: None Declared