OMICS Publishing Group | Full text | Pharmaco-Informatics: Homology Modelling of the Target Protein (GP1, 2) for Ebola Hemorrhagic Fever and Predicting an Ayurvedic Remediation of the Disease

Research Article	Open Access

Pharmaco-Informatics: Homology Modelling of the Target Protein
(GP1, 2) for Ebola Hemorrhagic Fever and Predicting an Ayurvedic Remediation of the Disease

Preenon Bagchi^*, Mahesh. M, Somashekhar.R

Azyme Biosciences, #1188/20, 3rd Floor, 26th Main, Jayanagar 9th Block, Bangalore-560069, India

^*Corresponding author:

Dr. Preenon Bagchi, Azyme Biosciences, #1188/20, 3rd Floor, 26th Main,
Jayanagar 9th Block, Bangalore-560069, India,
E-mail : preenonb@yahoo.com

Received February 24, 2009; Accepted April 15, 2009; Published July 07, 2009

Citation: Bagchi P, Mahesh M, Somashekhar R (2009) Pharmaco-Informatics: Homology Modelling of the Target Protein (GP1, 2) for Ebola Hemorrhagic Fever and Predicting an Ayurvedic Remediation of the Disease. J Proteomics Bioinform 2: 287-294. doi:10.4172/jpb.1000088

Copyright: © 2009 Bagchi P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Ebola hemorrhagic fever (Ebola HF) is caused by infection with Ebola Virus. Ebola virus, a member of the family Filoviridae, causes one of the most severe forms of viral hemorrhagic fever. In the final stages of the disease, symptoms progress to hypotension, coagulation disorders, and hemorrhages, and there is prominent involvement of the mononuclear phagocytic and reticulo-endothelial systems. It is assumed that the functions of the envelope glycoprotein are likely to play important roles in the pathogenicity of Ebola virus and the interactions of some viral proteins with the immune system are likely to play important roles in the extraordinary pathogenicity of this virus. Ebola virus (EBOV) entry requires the surface glycoprotein (GP) to initiate attachment and then fusion occurs between viral and host membranes. All glycoprotein forms are encoded by gene 4 of the EBOV genome. The strain selected is VGP_EBOSU with accession number Q7T9D9 of Sudan Ebola Virus - Uganda (2000) from NCBI’S entrez database. The 3D structure of Ebola Virus Protein was generated using Homology Modelling. For a predicted evaluation Andrographolide is used (the compound needs clinical trials to prove its efficacy in treatment). The 3D structure of Andrographolide was generated and was converted to *.pdb file which now docks with the *.pdb file of Ebola Virus Protein.

Keywords

Andrographolide; Andrographis panicula; Anti-Viral; Ayurveda; Ebola hemorrhagic fever; Ebola virus;
Filoviridae; Glycoprotein; Kalmegh; RNA viruses; Siddha; Zoonotic

Introduction

Ebola hemorrhagic fever (Ebola HF) is a severe, oftenfatal disease in humans and nonhuman primates (monkeys and chimpanzees) that has appeared sporadically since its initial recognition in 1976 [19]. All virions classified as hemorrhagic are enveloped (covered) RNA viruses, whose survival
is dependent on an animal reservoir. Viral hemorrhagic fever commonly describes a medical scenario in which multiple organ systems of the body are affected as well as extensive internal hemorrhaging (bleeding) The disease is caused by infection with Ebola Virus which is The World’s Deadliest Virus [24] (named after a river in the Democratic Republic of the Congo (formerly Zaire) in Africa, where it
was first recognized). The virus is one of two members of a family of RNA viruses called the Filoviridae [19] [24]. Three of the four subtypes of Ebola virus identified so far have caused disease in humans: Ebola-Zaire, Ebola-Sudan, and Ebola-Ivory Coast. The fourth, Ebola-Reston, has caused
disease in nonhuman primates, but not in humans [24]. The exact origin, locations, and natural habitat (known as the “natural reservoir”) of Ebola virus remain unknown. However, on the basis of available evidence and the nature of similar viruses, researchers believe that the virus is zoonotic (animal-borne) and is normally maintained in an animal host that is native to the African continent [19]. Ebola itself has an average length 920 nm and a diameter of 80 nm. The virus is considered a level 4 biohazard and is only handled in the most sterile environments in full protective suiting. Ebola
is spread through direct contact with blood or other bodily secretion of infected people (CDC, 2002; WHO, 2000).

Ebola envelope glycoproteins consist of a GP1 protein and membrane-bound GP2 protein that are covalently linked by a disulfide bond [Xin et al. (2005)] [Sanchez ., et al. (1996)]. Although the causes of filovirus virulence are not well defined, there is evidence that glycans on the viral glycoproteins play distinct roles in pathogenesis [Takada et al (2001)]. For example, expression of Ebola glycoprotein in cells causes a reduction in host cell-surface protein expression that is associated with cell rounding and detachment [Xin et al., (2005)] [Simmons et al., (2002)]
[Sullivan et al., (2005)]. Andrographis compounds have shown antivirus properties which appear to inhibit glycoprotein’s in the virus. This impedes the viruse’s ability to invade cells in the body and replicate. The signs and symptoms of Ebola HF are not the same for all patients. Symptoms characterizing Ebola are unspecific in the first few days of the infection, making the virus even more dangerous. Infection is marked by initial signs of fever, fatigue, exhaustion, muscle aches, and dizziness WHO, 2000. As the disease progress bleeding under the skin, in internal organs,
and from the eyes, ears, and mouth are seen. Patients with severe progressions of the disease express symptoms of shock, delirium, coma, seizures, and nervous system malfunction (CDC, 2002). Patients surviving infection by Ebola virions were found to develop stronger antibody responses in the early stages of infection than patients who eventually succumbed to the disease [Takada et al., (2001)]. The role of the innate immune response in the first few days of infection is considered very important in control of viral replication. Conversely up regulation of interluken 2, 10, tumor
necrosis factor, and interferons are associated with infection of the Ebola virus [Takada et al., (2001)]. Although their role is poorly understood, antibodies are thought to play an essential role in inhibiting infection of Ebola [Maruyama et al., (1999)]. Antibodies have been found that bind to
the nucleoprotein, the envelope protein, and the secreted envelope glycoprotein. Studies have shown that neutralizing antibodies made in response to these glycoproteins are effective against the Ebola virus and show some promise in designing a vaccine [Maruyama et al., (1999)]. The table below outlines symptoms of the disease, according to the frequency with which they have been reported in known cases [24].

Time Frame	Symptoms that occur in most Ebola patients	Symptoms that occur in some Ebola patients
Within a few days of becoming infected with the virus:	high fever, headache, muscle aches, stomach pain, fatigue, diarrhea	Sore throat, hiccups, rash, red and itchy eyes, vomiting blood, bloody diarrhea
Within one week of becoming infected with the virus:	chest pain, shock, and death	Blindness, bleeding

Known Epidemics

Known human cases and deaths during outbreaks of Sudan ebolavirus between 1976 and 2003 [29].

Sudan ebolavirus was the second strain of Ebola reported in 1976. It apparently originated amongst cotton factory workers in Nzara, Sudan. The first case reported was a worker exposed to a potential natural reservoir at the cotton factory. Scientists tested all animals and insects in response
to this, however none tested positive for the virus. The carrier is still unknown ES, 1976.

A second case involved a nightclub owner in Nzara, Sudan. The local hospital, Maridi, tested and attempted to treat the patient; however, nothing was successful, and he died. The hospital did not advocate safe and practical procedures in sterilizing and disinfecting the medical tools used on the nightclub owner, likely facilitating the spread of the virus in the hospital [29].

The most recent outbreak of Sudan ebolavirus occurred in May 2004. As of May 2004, 20 cases of Sudan ebolavirus were reported in Yambio County, Sudan, with five deaths resulting. The Centers for Disease Control and Prevention confirmed the virus a few days later [18] [30]. The neighbouring countries of Uganda and the Democratic Republic of Congo have increased surveillance in bordering
areas, and other similar measures have been taken to control the outbreak [29]. The average fatality rates for Sudan ebolavirus were 54% in 1976, 68% in 1979, and 53% in 2000/2001 [21].

Ayurveda (‘science of life’) is a system of traditional medicine native to India and practiced in other parts of the world as a form of alternative medicine. In Sanskrit, the word Ayurveda comprises the words ayus, meaning ‘life’ and veda, meaning ‘science’ [5] [23]. The earliest literature of Ayurveda appeared during the Vedic period in India. TheSushruta Samhita and the Charaka Samhita were influential works on traditional medicine during this era. Ayurvedic practitioners also identified a number of medicinal preparations and surgical procedures for curing various ailments and diseases Department of Ayurveda.

Andrographis paniculata, the Kalmegh of Ayurveda [23] is an erect annual herb extremely bitter in taste in each and every part of the plant body [27] [26]. When in bloom, Andrographis exhibits small white flowers [16] [28]. The plant is known in north-eastern India as ‘Maha-tita’, literally ‘king of bitters’. Scientists have studied this herb for nearly thirty years [25] [22]. Since ancient times, A. paniculata is used as a wonder drug in traditional Siddha and Ayurvedic systems of medicine as well as in tribal medicine in India and some other countries for multiple clinical applications. The therapeutic value of Kalmegh is due to its mechanism of action which is perhaps by enzyme induction. The splant extracts exhibits antityphoid and antifungal activities [22]. Kalmegh is also reported to possess antihepatotoxic, antibiotic, antimalarial, antihepatitic, antithrombogenic, antiinflammatory, antisnakevenom, and antipyretic properties to mention a few, besides its general
use as an immunostimulant agent. A recent study conducted at Bastyr University, USA confirms anti-HIV activity of andrographolide [22] [Calabrese et al., (2000)].

Andrographolide is a labdane diterpenoid that is the main bioactive component of the medicinal plant Andrographis paniculata [20] [Chakravarti et al., (1951)] [28]. Andrographolide is an extremely bitter substance extracted from the stem and leaves of the Kalmegh (Andrographis paniculata) [28].

Methodology

The following work utilizes the protocol involved in Computer Aided Drug Designing. The Ebola Virus selected is Sudan Ebola Virus - Uganda (2000).

Softwares/Web Servers used

1)	modeller9v5 MODELLER (copyright © 1989-2008 Andrej Sali) http://salilab.org/index.html {The most widely used software for homology (theoretical) modelling; also has option to take three templates as input for model generation}.
2)	Swiss-PdbViewer v4.01 by Nicolas Guex , Alexandre Diemand , Manuel C. Peitsch , & Torsten Schwede (Swiss Institute of Bioinformatics) http://spdbv.vital-it.ch/index.html {Here used for visualization, has option to provide distinct color to protein & molecule}.
3)	ACD/ChemSketch Freeware, version 11.00, Advanced Chemistry Development, Inc., Toronto, ON, Canada, www.acdlabs.com, 2008. {Widely used academic software for chemical structure drawing, software like hyperchem available}.
4)	RAMPAGE: Assessment of the Ramachandran Plot MolProbity \| Crystallography and Bioinformatics Group http://mordred.bioc.cam.ac.uk/~rapper/rampage.php {A trusted server for Ramachandran Plot analysis only to indentify the best protein generated by homology modelling in the article (also servers like VADAR, & softwares like SPDBV & PROCHECK can be used for the same purpose)} S.C. Lovell, I.W. Davis, W.B. Arendall III, P.I.W. de Bakker, J.M. Word, M.G. Prisant, J.S. Richardson and D.C. Richardson (2002) Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins: Structure, Function & Genetics. 50: 437-450.
5)	ArgusLab 4.0.1, Mark A. Thompson, Planaria Software LLC, Seattle, WA http://www.arguslab.com {particularly used for molecule format converter}.
6)	HEX_SERVER http://www.csd.abdn.ac.uk/hex_server/ {A good & trusted software for docking and free for academic usage} High Order Analytic Translation Matrix Elements For Real Space Six-Dimensional Polar Fourier Correlations, D.W. Ritchie (2005) J. Appl. Cryst. 38, 808-818.

The strain selected is VGP_EBOSU with accession number Q7T9D9 of Sudan Ebola Virus - Uganda (2000) from http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=protein&id=75559166.

The 3D (*.pdb) structures of VGP_EBOSU were generated by the process of Homology Modelling (using modeller 9v5) using the templates: 2EBOA (Identity=91%) (Source Strain: Ebola Virus Zaire Mayinga), 2RLJA (Identity=78%), (Source Stain: Zaire ebolavirus (ZEBOV) and 3CSYA (Identity=
64%) (Source: Zaire Ebola Virus Glycoprotein in complex with a neutralizing antibody from a Human Survivor of the 1995 Kikwit). The best templates were selected by submitting VGP_EBOSU protein coordinates to HHpred - Homology detection & structure prediction by HMM-HMM comparison @ http://toolkit.tuebingen.mpg.de/hhpred and to BLAST search engine. Five *.pdb structures was generated by modeler. These structures were submitted to Ramachandran Plot Server and best protein (*.pdb) was selected.

For treatment we have chosen a remedy from Indian traditional Ayurvedic practice. We have chosen Kalmegh (Andrographis paniculata) (based on ref. no.: 1 [a study by Ajoy et.al,] which determine the anti-viral property of Kalmegh and also from the recent study conducted at Bastyr University, USA confirming anti-HIV activity of Andrographolide [Calabrese et al., (2000)]).

The leaves contain the highest amount of andrographolide. It is the primary medicinal component of Kalmegh. It has a very bitter taste, is a colorless crystalline in appearance, and is called a “diterpene lactone” - a chemical name that describes its ringlike structure. Besides the related bitters cited above, other active components include 14-deoxy- 11,12- didehydroandrographolide (andrographlide D) [15].

The chemical structure of andrographolide was derived with ACD/Chemsketch software and is converted to *.pdb file (andrographolide.pdb) with the help of Arguslab software. Then finally it is found that VGP_EBOSU.pdb docks with andrographolide.pdb (by submitting the *.pdb files to hex server).

Results

The VGP_EBOSU.pdb protein obtained after homology modeling was analyzed with Rampage Ramachandran Plot server. The results obtained:

Model 1:

	Number of residues in favoured region (~98.0% expected): 634 (94.1%)
	Number of residues in allowed region (~2.0% expected): 26 (3.9%)
	Number of residues in outlier region : 14 (2.1%)

Model 2:

	Number of residues in favoured region (~98.0% expected): 627 (93.0%)
	Number of residues in allowed region (~2.0% expected): 37 (5.5%)
	Number of residues in outlier region : 10 (1.5%)

Model 3:

	Number of residues in favoured region (~98.0% expected): 635 (94.2%)
	Number of residues in allowed region (~2.0% expected): 30 (4.5%)
	Number of residues in outlier region : 9 (1.3%)

Model 4:

	Number of residues in favoured region (~98.0% expected): 634 (94.1%)
	Number of residues in allowed region (~2.0% expected): 32 (4.7%)
	Number of residues in outlier region : 8 (1.2%)

Model 5:

	Number of residues in favoured region (~98.0% expected): 628 (93.2%)
	Number of residues in allowed region (~2.0% expected): 32 (4.7%)
	Number of residues in outlier region : 14 (2.1%)

Model 4 was selected as the best model as per Ramachandran Plot analysis since it has least number of residues in outlier region.

Model 4 was found to dock successfully with Andrographolide.pdb.

Figure1: Sudan Ebola Virus.

Source: http://www.earthhopenetwork.net/Ebola_Outbreak_Suspected_in_Loss_of_Congo_Gorillas.htm

Figure2: Best modelled structure of Ebola Virus Protein (VGP_EBOSU.pdb) (Visualization in Rasmol).

Figure3: Ramachandran Plot of VGP_EBOSU.pdb (best model).

Figure4: Kalmegh

Source: http://www.biogreennutrachem.com/images/kalmegh.JPG

Figure5: Drawing of andrographolide structure using ACD/Chemsketch.

Figure6: Andrographolide.pdb (Visualization in Rasmol).

Figure7: Docked structure of VGP_EBOSU.pdb and andrographolide.pdb (visualization in SPDBV).

Conclusion

Since VGP_EBOSU.pdb was found to dock with andrographolide.pdb, it can be predicted that andrographolide can serve as a treatment for Ebola Hemmorrahagic Fever.

Future Perspectives

The above work is solely a Bioinformatics work developed using computational tools. Since without effective clinical studies, the compound can’t be considered as treatment for Ebola Hemorrhagic Fever. So the authors would carry out the trails in a virology lab to establish the effectiveness of andrographolide in treating Ebola Hemorrhagic Fever and concentration and the dosage of the compound.

Discussion

Since the above work is an in-silico work, the predicted compound (which proves its efficacy after docking studies) has to go for clinical trials to establish its effectiveness in curing the disease. The above work aims to serve all those researchers and patients in African Continent who are currently
experiencing this incurable disease.

The in-silico approach helps the researchers by giving them an in-hand idea so that they can happily advance towards the treatment of the disease. The in-silico-herbal work is presently an important subject of research since it is time saving, enables effective utilization of funds supplied and gives the best and well predicted results for effective utilization. Again, herbs have the least or no side effects and can be easily metabolized by the body. The study on virulence and extent to which this Ebola virus will infect human body was proved [11].

The anti-viral property of the drug was reported [31] and [Yi-Feng et al., (2004)]. In the present study the Indian Ayurvedic herb, kalmegh (Andrographis paniculata), is used. It has been used for medicinal purposes for centuries in India but the anti-viral property of this herb was unknown
to the world for over a decade. Also work aims to prove that no disease is incurable but the cure may be hidden in some other form. Also this work aims to highlight application of Bioinformatics in Drug Designing.

Dedication

This work is dedicated to Dr. Ajit Kar of Satsang Rasaisana Mandir (Satsang Chemical Works), Deoghar, who has followed the ideology and guidelines of Lord Sri Sri Thakur Anukulchandra and dedicated ayurveda to the service of mankind.

References

Basak A, Li S, Banik U (2003) A new combination drugs using andrographolide derived natural product Restomune for management of HIV. Case Rep Clin Pract Rev 4: 223-233.» CrossRef » Google Scholar
Calabrese C, Berman SH, Babish JG, Ma X, Shinto L, (2000) A phase I trial of andrographolide in HIV positive patients and normal volunteers. Phytotherapy Res 14: 333-338. » CrossRef » PubMed » Google Scholar
Center for Disease Control (2002) Ebola Hemorrhagic Fever. http://www.mindspring.com/~cinque/ebola.html
Chakravarti RN, Chakravarti D (1951) Andrographolide, the active constituent of Andrographis paniculata Nees; a preliminary communication”. Ind Med Gaz 86: 96-7. » PubMed » Google Scholar
Department of Ayurveda, Yoga & Naturopathy, Unani, Siddha and Homoeopathy. Ministry of Health & Family Welfare [Government of India].
Ebola hemorrhagic fever in Sudan, 1976, Bulletin of the World Health Organization. 56: 247-270.
Maruyama T, Rodriguez LL, Jahrling PB, Sanchez A, Khan AS, (1999) Ebola virus can be effectively neutralized by antibody produced in natural human infection. Journal of Virology 73: 6024-30. » CrossRef » PubMed » Google Scholar
Sanchez A, Trappier SG, Mahy BW, Peters CJ, Nichol ST (1996) The virion glycoproteins of Ebola viruses are encoded in two reading frames and are expressed through transcriptional editing. Proc Natl Acad Sci USA 93: 3602-3607. » CrossRef » PubMed » Google Scholar
Simmons G, Wool-Lewis RJ, Baribaud F, Netter RC, Bates P (2002) Ebola virus glycoproteins induce global surface protein down-modulation and loss of cell adherence. J Virol 76: 2518-2528. » CrossRef » PubMed » Google Scholar
Sullivan NJ, Peterson M, Yang ZY, Kong WP, Duckers H, (2005) Ebola virus glycoprotein toxicity is mediated by a dynamin-dependent protein-trafficking pathway. J Virol 79: 547-553. » CrossRef » PubMed » Google Scholar
Takada A and Kawaoka Y (2001) The pathogenesis of Ebola hemorrhagic fever. Trends in Microbiology 506: 108-8639. » CrossRef » PubMed » Google Scholar
Xin J, Olinger GG, Aris S, Chen Y, Gewurz H, (2005) Mannose-binding lectin binds to Ebola and Marburg envelope glycoproteins, resulting in blocking of virus interaction with DC-SIGN and complementmediated virus neutralization. J Gen Virol 86: 2535-2542. » CrossRef » PubMed » Google Scholar
Xia YF, Ye BQ, Li YD, Wang JG, He XJ, (2004) Andrographolide Attenuates Inflammation by Inhibition of NF-B Activation through Covalent Modification of Reduced Cysteine 62 of p50. J Immunol 173: 4207-4217. » CrossRef » PubMed » Google Scholar
World Health Organization (2000) Ebola Hemorrhagic Fever.
http://www.who.int/inf-fs/en/fact103.html

URL References

This Article

DOWNLOAD

» XML (39 KB)
» PDF (836 KB)
» Citation

CONTRIBUTE

» Write a Response
» Read other Responses
» Publishing with OMICS
Publishing Group

» E-mail This Article
» Print This Article
» Rights and Permissions

EXPLORE

Related Article at

» Pubmed
» DOAJ
» Scholar Google

Perspectives

Abstract
Introduction
Methodology
Results
Conclusion
Discussion
References