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Crimean Congo Hemorrhagic fever
- 1. A Computational Approach to Explore Crimean Congo Hemorrhagic Fever PRESENTED BY: ALICHY SOWMYA REG. NO. 18PIM2855 M.S.(PHARM.) 1ST SEM. DEPARTMENT OF PHARMACOINFORMATICS NIPER, S.A.S NAGAR
- 3. • Crimean Congo Hemorrhagic Fever is a disease caused by an Arbovirus, CCHFV with a mortality rate of 6% to 50% • The first description of hemorrhagic fever similar to that of CCHF was done in 12th century • Officially it was first described, in 1940s in the Crimean peninsula, and later in 1956 in Congo • It is geographically widespread, ranging from the Middle East and Asia to South Africa and Eastern Europe • CCHFV is biosafety level-4 virus making it difficult to study Introduction: 2 Mendoza, E., et al.,, Zoonoses Public Health. 2018, 65, 465-480.
- 4. Epidemiology: • The disease is endemic in many countries in Africa, Europe and Asia, and outbreaks have been recorded in Kosovo, Albania, Islamic Republic of Iran, Pakistan, India, and South Africa among other countries • In between 2002 – 2008 , 2508 cases of CCHF with 133 deaths have been reported in Turkey alone • In September, 2010 an outbreak was reported in Pakistan with a case-fatality rate above 10% • In SEAR, the first laboratory confirmed case was reported on 19 January, 2011 in Gujarat state of India 3 http://www.searo.who.int/entity/emerging_diseases/links/CCHF_Fact_Sheet_SEARO.pdf (21 nov 10:30am).
- 5. Scenario in India: • In India first case of CCHF was reported in Jan 2011, in Gujarat, then there was a nosocomial outbreak talking 3 lives • Again in 2013, there was a outbreak in Karyana village, Amreli district, Gujarat State • CCHF-positive and fatal human cases were reported from Patan, Rajkot, Surendranagar, and Ahmadabad districts in Gujarat State • In may 2018, CCHF was reported in Sirohi district, Rajasthan state Mishra et al., Lancet. 2011, 378, 372. 4
- 6. Causative agent: • CCHFV is a second most widely distributed arbovirus after dengue virus • Classification: • Virion structure and molecular biology: Flick, R.et al., Curr. Mol. Med. 2005, 5, 753-760. Contd… 5
- 7. • Transmission of virus: Small Mammals and birds Nymph Larva Drop off and Molt Oviposition and Hatching Adult Humans (Nosocomial transmissions) Contd… 6 Aslam, S., et al.,, Biomed Rep. 2016, 4, 15-20.
- 8. Pathophysiology: CCHFV Tick bite Entry from epithelium Basolateral compartment Release in the endothelial cells Basolateral compartment Spread into the blood stream Dissemination into liver and other tissues Release of cytokines Upregulation of the adhesion molecules Damage of the immune system Necrosis with little or no inflammation Activation of Endothelial cells and damage of the endothelium Uncontrolled viral replication Initiation of the inflammatory reactions Intrinsic coagulation cascade activated Vascular permeability and vasodilation are increased Disseminated intravascular coagulation Hypotension, multiple organ failure and shockHemorrhages Liver dysfunction Hemophagocytosis CCHFV Infected dendritic cells and Macrophages Spread to local lymph nodes and spleen 7 Arasli, M., Curr Trop Med Rep. 2016, 3, 14-19.
- 9. Clinical Features: • The CCHFV infection can be characterised by four characteristic stages viz., incubation, prehemorrhagic, hemorrhagic, and convalescence Melena Epistaxis Ecchymosis Petechiae Ergönül, Ö.et al., Emerg. Infect. Dis. 2018, 24, 1642. 8
- 10. Treatment: • CCHF is a severe disease in humans, with a high mortality rate, but unfortunately there is no specific treatment available till date • General supportive therapy is the mainstay of patient management in CCHF. Intensive monitoring to guide volume and blood component replacement is required • The antiviral drug ribavirin has been used in treatment of established CCHF infection with apparent benefit, but effectiveness is yet to be proven 9 http://www.searo.who.int/entity/emerging_diseases/links/CCHF_Fact_Sheet_SEARO.pdf (21 nov 10:30am).
- 11. Prevention and control: • As it is a tick borne virus acaricides (chemicals intended to kill ticks) can be an option • Persons living in endemic areas should use personal protective measures • Patients with suspected or confirmed CCHF should be isolated and cared for using barrier nursing techniques. Proper measures are to be taken to prevent nosocomial transmission • Healthcare workers are at risk of acquiring infection and therefore they should take proper precautions • Community education initiatives should be instituted in the event of an observed outbreak 10 http://www.searo.who.int/entity/emerging_diseases/links/CCHF_Fact_Sheet_SEARO.pdf (21 nov 10:30am).
- 12. Literature review: 11 • Javadmanesh Ali et al., have conducted in silico evaluation of the antiviral effects of the lactoferrin against the envelopment protein (Gn and Gc) of the CCHFV • Sitansu Kumar Verma et al., have conducting docking studies to explore the binding sites for the antiviral compounds in RdRp of CCHFV • Fatih Kocabas et al., used computational approaches for identification of inhibition site in OTU Domain of CCHFV
- 14. • Materials and methods: Preparation of the target structure for docking Preparation of the docked entity Predicting CCHFV N binding site High – throughput docking studies Classical Molecular dynamic simulation Ensemble – based docking simulations Virtual screening procedure • Results: CCHFV N druggable pocket Finding putative inhibitors Molecular dynamic studies Contd… Contd… 13
- 15. Contd… Contd… Preparation of the target structure for docking: •The X Ray crystallographic structure of CCHFV nucleoprotein was deposited in the PDB along with sulfate ion as a ligand with ID 4AQG with a resolution of 2.8 Å •They have downloaded the structure and upon studying they have found that the amino acid sequence was missing of 12 amino acid sequences which were later added using SWISS - MODEL •The sequences from Leu181 to Ser194 were added 14
- 16. Contd… Contd… Preparation of the docked entity: 15
- 17. Contd… Contd… Predicting CCHFV N binding site: •In this study for determining the binding pocket, they have used DoGsiteScorer server •Then they have used Poisson Boltzmann solver (APBS) method of PyMol software to determine the positive charges High – throughput docking studies: •After the binding pockets have been recognized they have predicted the binding poses and affinities of the compounds over the predicted binding site of CCHFV Nucleoprotein •For this determination AutoDock Vina 1.1.2 with 40 – 34 – 40 search space was used 16
- 18. Classical Molecular dynamic simulation: •Initially, they have used PRODRG server to generate the topology files of the ligand •The corrections regarding their protonation states was made using the PROPKA server and finally GROMAC was used •The molecular dynamics simulation was done for 10 ns Ensemble – based docking simulations: •In this study, the ensemble was made using molecular dynamic simulation •The simulation was performed for 10 nanoseconds (ns) and for every 2.5 ns a snapshot was taken Contd… Contd… 17
- 20. Contd… Contd… Results: CCHFV N druggable pocket: •Using DoGsiteScorer server they have recognised three pockets which are allegedly be the potential binding pocket. The three pockets were as follows: • Pocket1 (between head and tail domains) • Pocket 2 (in the head domain) • Pocket 3 (in the head domain) •It was found that pocket 1 and pocket 3 consists of both positive and negative charges, and the pocket 2 contains more positive charge 19
- 21. Contd… Contd…Sharifi. A et al., Comput. Biol. Chem. 2017, 70, 1-6. Three predicted pockets over CCHFV N protein are coloured by yellow, cyan and magenta 20
- 22. Contd… Contd… Finding putative inhibitors: •1584 FDA approved drugs were docked with the cut-off value of -8 kcal/mol and 25 hits were retained •After the screening procedure only Doxycycline and Minocycline have been selected •The docking conformation of doxycycline shows two hydrogen bonds with Leu387 and one with Lys411 besides hydrophobic interactions with Ala302, Ile373, and Ile448 •Minocycline predicted pose comprises only one hydrogen bond with Ser386 in addition to hydrophobic contact with Ala302, Thr381, and Ile448 residues •It was hypothesized that doxycycline inhibits the protein more desirable than minocycline, as it is in accordance with their obtained binding energies 21
- 23. Contd… Contd… A. Binding orientation of doxycycline B. Binding orientation of minocycline C. Alignment of both drugs 22 Sharifi. A et al., Comput. Biol. Chem. 2017, 70, 1-6.
- 24. Contd… Molecular dynamic studies: •The Root Mean Square Deviation (RMSD) of the protein backbone reveals that the suggested ligand – protein complex reach stability at same time (A) MD graphs of doxycycline and minocycline (B) The RMSD plot of protein ligand complex 23 Sharifi. A et al., Comput. Biol. Chem. 2017, 70, 1-6.
- 25. Conclusion: • CCHFV is one of the most dangerous classes of the virus and it was suggested CCHFV Nucleoprotein could be a potential pharmaceutical target for inhibiting CCHFV • FDA approved drugs were docked 25 dugs were filtered, virtual screening of these 25 drugs were conducted and two drugs, namely Doxycycline and Minocycline were selected • Molecular dynamic simulation of CCHFV N protein was done for 10ns with a snapshot taken for every 2.5ns i.e., 4 snapshots or 4 conformations of the protein were identified and the two drugs Doxycycline and Minocycline were docked over those 4 conformers • It was found that both bind to all conformers with equal affinity and RMSD revealed that the protein-ligand complex is more stable than that of protein without ligand 24