J Bio Life Sci 2011 2(1):5-10

Journal of Biology & Life Sciences

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Original Article

Insilico Identification of Novel Inhibitors for Emphysema through Structure Based Drug Design Approaches
Raghavan RAJASHEKARAN1, Charanya MURALIDHARAN2, Sathyabaarthi RAVICHANDRAN2 Umashankar VETRIVEL2*
2

Birla Institute of Technology and Science, Pilani Goa Campus, Goa, India Center for Bioinformatics, Vision Research Foundation, Sankara Nethralaya, Chennai, India Received: 15.11.2010 Accepted: 12.12.2010 Published: 18.12.2010

Abstract Emphysema is a Chronic Obstructive Pulmonary Disease (COPD), which results due to imbalance in levels of elastase and its endogenous inhibitors. Decreased level of Human Neutrophil Elastase (HNE) inhibitors owes to the tissue destruction and structural disorientation of alveoli. Current treatment procedures involves intravenous administration of Alpha 1 Anti Trypsin (A1AT) to potentially control the level of HNE .Recent studies show decreased efficacy of A1AT due to non adaptive pharmacodynamics properties of lung. This study involves Insilco identification of potential small molecule inhibitors by implementing protein- protein interaction analysis and pertaining to potential lead identification. The structural analogues of the lead compound (ligand of Coagulation Factor VII) namely, CID23646455, CID6102769 and CID10224813 were found to show strong interaction with HNE in comparison to the experimentally verified inhibitors and other analogues with a binding energy of -9.08081 Kcal/mol, -9.0617 Kcal/mol and -9.01617 Kcal/mol, respectively. Hence, the identified small molecules shall prove to be a potential entity in modulating this disease manifestation.
Key words: Emphysema, HNE, A1AT, Pharmocophore, Docking *Corresponding Author: Dr. V. Umashankar, e-mail: drvus@snmail.org, Phone: 044-28270709, Fax: 044-28254180

INTRODUCTION Chronic Obstructive Pulmonary Disorder (COPD) is one of the leading death causing disease worldwide(Rabe et al. 2007). Bronchitis, bronchiolitis and emphysema, are few diseased conditions which fall under COPD category(Petty 1998). Emphysema is the condition in which the damage in lung tissue leads to structural disorientation of alveoli, which in turn affects the normal functional properties. Emphysema affects 40% of heavy smokers and causes loss of elastic recoiling property, leading to abnormal gas exchange and breathlessness(Silvers et al. 1980; Baldi et al. 2001). The death ratio of pulmonary emphysema was found to be 100,000 deaths/year on an average (http://www.nhlbi.nih.gov/health/prof/lung/nett/lvrsweb.htm). Alpha 1 Anti Trypsin (A1AT) is a serine protease inhibitor produced in liver which potentially inhibits Human Neutrophil Elastase (HNE). A1AT deficiency leading to lack of HNE inhibition is the major reason for emphysema progression. This in turn leads to inflammation, protease-antiprotease imbalance and oxidative stress conditions(Petrache et al. 2006). Mutations of A1AT gene results in decreased concentration and proteolytic dysfunction of A1AT protein(Crowther et al. 2004). In normal conditions, A1AT inhibits elastase, which in turn regulates elastic recoiling property of the lungs by maintaining the levels of elastin. Hence, impaired A1AT function results in lack of elastase inhibition resulting in loss of lung elasticity due to the absence of elastin breakdown (Thorley and Tetley 2007). Current treatment strategy of intravenous administration of A1AT ameliorates smoke induced inflammation and matrix breakdown through anti-inflammatory mechanism of tumor necrosis factor and it controls emphysema to a very less extent. However, current treatment doesnt completely

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control the severity of the emphysema despite of many research outcomes(Churg et al. 2003). Many of the reported small molecular inhibitors of HNE failed due to undesirable pharmacodynamic properties that are not adaptable to lung physiology. Hence, in this study, it is hypothesized to identify the potential small molecular inhibitor(s) which mimics the function of A1AT using structural bioinformatics approach.

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minimisation using Universal Force Field (UFF) (da Silva et al. 2006). Docking simulation studies between HNE and the structurally optimized ligands were carried out using Argus Lab (Stoermer 2006). Hydrogen bonding and hydrophobic interactions of the ligands with HNE were analyzed and visualised using Discovery studio visualizer 19 (Talley et al. 2008) and PyMOL (Wijeyesakere et al. 2007).

MATERIAL AND METHODS Sequence Conservation Analysis The sequence information of ATA1 (P01009) and HNE (P08246) were retrieved from Uniprot Knowledgebase (http://www.uniprot.org/uniprot). In order to identify functionally significant residues (based on conservation), the protein sequences of ATA1 and HNE were compared with its close homologues using ConSurf (Ashkenazy et al. 2010). Protein-Protein docking The atomic co-ordinates of HNE (PDB ID: 2Z7F) and A1AT (PDB ID: 2QUG) were retrieved from Protein Data Bank (http://www.pdb.org/pdb/home/home.do). Cluspro, an online server, which uses rigid protein-protein docking methodology was used to predict the interactions between HNE and A1AT (Kozakov et al. 2010). The residues involved in hydrogen bonding and hydrophobic interactions of the resultant A1AT-HNE complex were analyzed using HBPLUS algorithm provided in PDBsum (Laskowski 2009). The identified binding site residues of the docked complex were analyzed for interactions using PyMol (Wijeyesakere et al. 2007). Lead Identification The Proteins which shared similar pharmacophore to that of HNE (in terms of volume, shape and residues involved) were identified using SUMO(Jambon et al. 2003), an online server for identification of macromolecules with similar binding pockets. The small molecule which was bound to the protein that shared significant similarity with the binding cavity of HNE was selected as the lead compound. The structural data of the lead compound was retrieved from PubChem database (Wang et al. 2009). Virtual screening of compounds similar to the identified lead was searched against PubChem database via VEGA ZZ(Pedretti et al. 2004). Lipinski rule of five was implemented to remove the compounds which had less possibility of being a drug (Walters et al. 1999). Protein-ligand docking Furthermore, geometry optimization of the identified structural analogues and the lead were performed by energy

RESULTS AND DISCUSION Conservation Scoring of HNE and A1AT Functionally conserved residues of HNE in comparison to Serine Protease were mapped based on the conservational score as predicted by ConSurf analysis (Fig.1A). Similarly for A1AT, functionally significant residues were mapped by comparing with the Serine Protease inhibitor family. The residues mapped were suggestive of structural and functional importance (Fig.1B). Docking Simulation of HNE with A1AT Docking simulation studies were performed for HNE with A1AT using ClusPro server which works on the basis of molecular shape complementarity (Fig. 2A).Docking interaction studies of HNE with A1AT reveals the involvement of biologically significant residues predicted through ConSurf Analysis. PDBsum interaction analysis predict that Arg196, Asp280,Arg281, Arg282, Leu353 and Ser359 of A1AT were found to interact with Tyr96, Val99, Asn100, Arg207,T yr215, Gly208, and Cys210 of HNE, respectively. (Table 1) (Fig. 2B). Based on the interacting residues, the binding cavity of HNE was mapped(Fig. 2A).The residues interacting between HNE with A1AT were visualised using Discovery Studio visualiser and tabulated (Fig. 2B)(Table 1). Based on the identified interacting residues, binding pocket of HNE was mapped (Fig. 2A) and similar binding cavities were identified using SUMO (Fig.2).
Table 1 Hydrogen bonding interaction between HNE and A1AT as predicted by PDBsum Interacting residues in HNE Arg196 Arg281 Asp280 Arg282 Arg282 Leu353 Ser359 Interacting residues in A1AT Gly208 Tyr215 Arg207 Val99 Asn100 Cys210 Tyr96 Hydrogen bonding Distance in () 2.62 2.57 2.66 2.57 3.23 2.79 2.74

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Figure 1 ConSurf Results A) ConSurf Result of HNE. (B)ConSurf Result of A1AT.(C). legend for ConSurf representation

Identification of Lead Compound The binding cavity mapped based on the interaction analysis was screened against the PDB using SUMO, to find the proteins that share similar structural scaffold. Blood Coagulation Factor VII (2BZ6), which is also a serine protease was found to be a significant match. Hence, (R)-(4CARBAMIMIDOYL-PHENYLAMINO)-[5-ETHOXY-2FLUORO-3-[(R)-TETRAHYDRO-FURAN-3-YLOXY]PHENYL]-ACETIC ACID (ligand 346), an inhibitor of Blood Coagulation Factor was chosen as a lead and the binding efficacy of HNE with the chosen lead was evaluated through docking interaction studies. The results showed that

the identified Lead has strong hydrogen bonding interactions with N196 and G197 of HNE (Fig. 4) with a binding energy of -8.08839Kcal/mol. This is suggestive of the drug repurposability of the identified lead molecule. Furthermore, the lead molecule was screened against PUBCHEM through VEGA ZZ to fetch significant structural analogues. The potential structural analogues which follows Lipinski rule were chosen and optimised to derive its near native confirmation by energy minimisation using UFF force field. The optimised three dimensional structures of the structural analogues with the control Heparin was shown in (Fig. 3).

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Figure 2 HNE-A1AT docking using Cluspro.(A) Ribbon representation of the docked complex of HNE (cyan) with A1AT (green) and the interacting regions were represented as surface.(B) Docking interaction analysis of HNE-A1AT using DS visualiser.

Figure 3 Three dimensional (3D) structures of screened compounds and Control Heparin

Table 2 Docking interaction Analysis: Binding energy of the complex with the hydrogen bonding and non bonded interacting residues of HNE with the ligands SI.no Lead Ligand Sumo predicted Control Inhibitors Ligand 346 Heparin Binding Energy (Kcal/mol) -8.08839 -8.9134 Hydrogen bonds N196 and G197 V94, W143, G72, S 189, G142, W27, Q 157, A73 and M141 R20 and G197 Hydrophobic interactions Q137, R20, W27, Q158, V194, L 139, F29, N160 and C195 A26, H25, F219, H74, P28, F219, P192, V 161, T175, A203, T 175, M30, L156, L71, V155 A26, W27,P28,F29,Q137,C138 L139,F158,N160,V161,C195,V194 N196 and G197 L101, V184, C185, F186, A203,V206, F205, C210 and A218 V17 and L146 L101, V 184, C185, F186, F205, V206 and C210 C42, C58, L101, L145, V184, C185, F186, A203, F205 and V206 L101, V184, C185, F186, A203, F205, V206 F29, M141, C138, L139, V194, C195 V99, L101, L102, L167, C168, C174, A 218, F 205 H57,G187,S189,S204,V184,C185,F186, L101,F205,V206,R207,R169,N100,C210 F41,H57,V206,F205,A203,V184,C185,L 145,G187,D188,S189,,G190,S204,F184.

1.

CID6102769

-9.0617

2. 3. 4. 5. 6. 7. 8. 9. 10.

CID22039118 CID18551829 CID23646455 CID44405420 CID 9907633 CID10114609 CID10224813 CID22039106 CID6852220

-8.45933 -8.78736 -9.08081 -8.28867 -8.14015 -8.80311 -9.01617 -7.94356 -8.31548

V206, G208 and G209 G18, R21, R150 and G157 V206 and S189 H57 and S189 G187, S189, S204 and V206 N196 and G197 V99 and R169 H57, S189 and V206 --

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Docking studies of HNE with the identified structural analogues Protein-ligand docking of HNE was carried out with Heparin, a known inhibitor of HNE to serve as a control measure (Spencer et al. 2006). Molecular Docking between HNE and Heparin was performed and the resulting binding energy and interactions were tabulated (Table 2). Similarly, HNE was docked with other structural analogues and the results were tabulated (Table 2)(Fig.5). The potential structural analogues namely, CID6102769 and CID23646455 were found to show strong inhibition in comparison with the control in terms of binding energy, as well as in molecular

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interactions. CID6102769 showed a binding energy of 9.0617Kcal/mol with hydrogen bonding interactions to Arg20 and Gly197 of HNE. In case of CID23646455, it forms hydrogen bonding interaction with SER189 and VAL 206 with a binding energy of -9.08081 Kcal/mol. Docking analysis of CID10224813 revealed that it interacts with V99 and R169 of HNE with a binding energy of -9.01617 Kcal/mol. Furthermore, super positioning of CID6102769, CID23646455 and CID10224813 docked poses with that of lead ligand 346 and the control heparin were found to share same binding region, thereby following similar mechanism of action (Fig. 5).

Figure 4 Docking interactions shown using DS visualiser between HNE and screened ligands (represented in green)

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Baldi S, Miniati M, Bellina CR, Battolla L, Catapano G, Begliomini E, Giustini D, Giuntini C ,2001. Relationship between extent of pulmonary emphysema by high-resolution computed tomography and lung elastic recoil in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 164: 585-589 Churg A, Wang RD, Xie C, Wright JL, 2003. alpha-1-Antitrypsin ameliorates cigarette smoke-induced emphysema in the mouse. Am J Respir Crit Care Med 168: 199-207 Crowther DC, Belorgey D, Miranda E, Kinghorn KJ, Sharp LK, Lomas DA, 2004 Practical genetics: alpha-1-antitrypsin deficiency and the serpinopathies. Eur J Hum Genet 12: 167-172 da Silva CH, Campo VL, Carvalho I , Taft CA, 2006. Molecular modeling, docking and ADMET studies applied to the design of a novel hybrid for treatment of Alzheimer's disease. J Mol Graph Model 25: 169-175 Guarnieri F, Spencer JL, Lucey EC, Nugent MA, Stone PJ, 2010. A human surfactant peptide-elastase inhibitor construct as a treatment for emphysema. Proceedings of the National Academy of Sciences of the United States of America 107: 10661-10666 Jambon M, Imberty A, Deleage G , Geourjon C, 2003. A new bioinformatic approach to detect common 3D sites in protein structures. Proteins 52: 137-145 Kozakov D, Hall DR, Beglov D, Brenke R, Comeau SR, Shen Y, Li K, Zheng J, Vakili P, Paschalidis I, Vajda S, 2010. Achieving reliability and high accuracy in automated protein docking: ClusPro, PIPER, SDU, and stability analysis in CAPRI rounds 13-19. Proteins 78: 3124-3130. Laskowski RA , 2009. PDBsum new things. Nucleic Acids Res 37: D355359 Pedretti A, Villa L, Vistoli G, 2004. VEGA--an open platform to develop chemo-bio-informatics applications, using plug-in architecture and script programming. J Comput Aided Mol Des 18: 167-173 Petrache I, Fijalkowska I, Medler TR, Skirball J, Cruz P, Zhen L, Petrache H I, Flotte T R, Tuder RM, 2006. alpha-1 antitrypsin inhibits caspase-3 activity, preventing lung endothelial cell apoptosis. Am J Pathol 169: 1155-1166 Petty TL, 1998. Definitions, causes, course, and prognosis of chronic obstructive pulmonary disease. Respir Care Clin N Am 4: 345-358, vii. Rabe KF, Hurd S, Anzueto A, Barnes P J, Buist SA, Calverley P, Fukuchi Y, Jenkins C, Rodriguez-Roisin R, van Weel C, Zielinski J,2007. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 176: 532-555 Silvers GW, Petty TL, Stanford RE, 1980. Elastic recoil changes in early emphysema. Thorax 35: 490-495 Spencer JL, Stone PJ, Nugent MA, 2006. New insights into the inhibition of human neutrophil elastase by heparin. Biochemistry 45: 9104-9120. Stoermer MJ , 2006. Current status of virtual screening as analysed by target class. Med Chem 2: 89-112 Talley TT, Harel M, Hibbs RE, Radic Z, Tomizawa M, Casida JE, Taylor P, 2008. Atomic interactions of neonicotinoid agonists with AChBP: molecular recognition of the distinctive electronegative pharmacophore. Proceedings of the National Academy of Sciences of the United States of America 105: 7606-7611 Thorley AJ, Tetley TD, 2007. Pulmonary epithelium, cigarette smoke, and chronic obstructive pulmonary disease. Int J Chron Obstruct Pulmon Dis 2: 409-428 Walters WP, Ajay, Murcko MA, 1999. Recognizing molecules with drug-like properties. Curr Opin Chem Biol 3: 384-387 Wang Y, Xiao J, Suzek TO, Zhang J, Wang J, Bryant SH ,2009. PubChem: a public information system for analyzing bioactivities of small molecules. Nucleic Acids Res 37: W623-633 Wijeyesakere SJ, Richardson RJ, Stuckey JA .2007. Modeling the tertiary structure of the patatin domain of neuropathy target esterase. The protein journal 26: 165-172

Figure 5 Super Positioning of ligand 346, Heparin (control) and the most potent structural analogues. HNE is represented in surface in Yellow with A1AT binding site in blue. The ligands are represented in stick model. Heparin (Control) in cyan. Lead ligand- 346 in magenta. Potential ligands CID23646455, CID6102769 and CID10224813 are shown in green, red and orange, respectively

CONCLUSION In this present study, the results of docking analysis reveal that, CID6102769, CID 23646455 and CID10224813 were found to be more efficient inhibitors of HNE based on the binding energy and the strength of interactions in comparison to experimentally proven inhibitor, Heparin. CID 6102769, CID 23646455 and CID10224813 were found to show significant interaction with HNE with a binding energy of -9.0617Kcal/mol,-9.08081 Kcal/mol and -9.01617 Kcal/mol, respectively. Whereas, in case of Heparin (control), the binding energy was -8.9134 Kcal/mol. Furthermore, CID 6102769 shows strong hydrogen bonding interaction with Gly197 and Arg20, whilst, CID 23646455 showed strong interactions with Ser189 and Val206. CID10224813 formed hydrogen bonding interaction with Val99 and Arg169. All these reported drug binding residues were found to be functionally significant and are involved in HNE activity, as per ConSurf prediction. Hence, the identified small molecular inhibitors can be further validated for its invivo efficacy and pharmacokinetics. Moreover, these ligands can also be covalently linked to the existing peptide construct to enhance the bioavailability in lungs (Guarnieri et al. 2010). Hence, by this study we propose few of the novel inhibitors which can used for modulation of Emphysema disease manifestation. REFERENCES
Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N, 2010. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res 38 Suppl: W529-533

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