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

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.


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 J Proteomics Bioinform Volume 2(7) : 287-294 (2009) -288 ISSN:0974-276X JPB, an open access journal (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). 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).

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

Softwares/Web Servers used
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:

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 devel-

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 (Takada and Kawaoka, 2001).
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.