The dengue virus structure

A presentation on dengue virus structure, how the virus attacks and spreads in the body, role of heterocyclic drugs in inhibiting the virus and our experiments on the subject.

1. Screening of heterocyclic drugs for its adaptability in inhibiting dengue virus MINI-PROJECT PRESENTATION BY JAMES ISSAC-(1RV10CH010) MUSTAFA HARIS-(1RV10CH017) S. GAUTHAM-(1RV10CH027) JACOB JOHN- (1RV10CH042)

2. DRUG DISCOVERY METHODS High throughput screening Virtual screening Scaffold hopping Rational design- hand built drugs Natural products HITS DRUG DISCOVERY Drug Development Hit Success rate:<1% Clinical Trials success rate: 20% NEW DRUG!

3. The Dengue fever Dengue virus (DENV) belongs to the Flavivirus genus of the Flaviviridae family. Transmitted by Aedes Aegypti . The Diseases : Dengue fever Dengue hemorrhagic fever (DHF) Dengue shock syndrome Threats : 50 million infections per year 500,000 develop DHF 22000 deaths per year Current Treatment : No vaccine Limited efficiency of antiviral drugs Treatment for DHF is blood transfusion

4. DENV 4 serotypes of the virus: DENV 1 to DENV 4 Its genome comprises of a 10.7 kb single positive stranded RNA. Each serotype provides specific lifetime immunity, and short-term cross-immunity All serotypes can cause severe and fatal disease Genetic variation within serotypes most prevalent of the four dengue serotypes is Dengue virus type 2 (Den2) Single stranded RNA

5. The Dengue virus structure Carries a single stranded RNA as its genome. The genome encodes only 10 proteins. Out of which 3 are structural proteins and 7 are non structural proteins. The 3 structural proteins (C. prM and E) form the coat of the virus and deliver the RNA to target cells. The seven non structural proteins ( NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5) known as NS proteins. They orchestrate the production of new viruses once the virus gets inside the cell. 180 identical copies of envelope (E) protein are attached to the membrane by Transmembrane Segment. Envelope protein attaches to the cell surface and begins the process of infection.

6. The Protein Data Bank (PDB) is the single worldwide repository for processing and distribution of 3D structure data of large molecules of proteins and nucleic acids. The outermost structural protein termed as the envelope protein – from the PDB entry 1k4r.

7. EXPLORING THE STRUCTURE… Cryoelectron microscopy is used to study the life cycle of dengue virus. Low resolution image of the DENV obtained using electron microscopy. Atomic structures of individual pieces are fit into the image to form the final model. The image shown above is from PDB entry 2r6p. The envelope protein on the surface of the protein is shown(in white) The antibodies bound to the viral protein is shown (in blue) Antibodies distort the structure of the envelope proteins hence blocking their normal action in infection.

8. The DENV (+) RNA genome and its co-linear polyprotein NS3 protein from PDB entry 2vbc NS5 protein from PDB entry 1l97 and 2j7w Protease Helicase Methyl transferase Polymerase

9. Mechanism of action of the virus in the Body…. In the infectious form of the virus, the envelope protein lays flat on the surface forming a smooth icosahedral symmetry. The virus is carried into the cell and into the lysosome. Organelles: 1.Nucleolus 2.Nucleus 3. ribosomes ( little dots) 4. vescicle 5. Rough endoplasmic reticulum 6. Golgi appartus 7. Cytoskeleton 8. Smooth endoplasmic reticulum 9. Mitochondria 10. Vacuol 11. Cytosol 12. Lysosome 13. Centrriol 14. cell membrane

10. Inside the Lysosome , the acidic environment causes the protein to snap to a trimeric shape. Several hydrophobic amino acids at the tip of the spike pierce into the lyzosomal membrane . They fuse the virus membrane with the lysosomal membrane. Thus releasing the viral RNA into the cytoplasm of the cell and infection starts. Now the proteins in the virus help in replication NS3 and NS5 are two multifunctional proteins containing 2 enzymes each. NS3 from PDB entry 2vbc, contains a Helicase and Protease enzymes. NS5 from PDB entry 1l9k and 2j7w contains Methyl Transferase and Polymerase enzymes.

11. NS3 protein from PDB entry 2vbc Protease Helicase NS5 protein from PDB entry 1l97 and 2j7w Methyl transferase Polymerase Each of these enzymes perform a different part of the life cycle The Polymerase builds new RNA strands based on viral RNA. Helicase Helps to separate these strands. Methyl Transferase adds a methyl group to the end of them to protect the RNA and coaxing the Ribosome to create new proteins based on them. Viral proteins are created in one long polyprotein chain and are finally clipped together by the enzyme protease. Blue colour seen is the part of another protein NS2B which assists the protease activity.

12. ANTIVIRAL TARGETS The virus encoded proteins enzymes constitute the potential targets. Envelope , capsid , helicase , polymerase, proteases, etc The M and E proteins are considered as targets so far. The E protein is the most obvious target for therapeutic monoclonal antibodies. Out of the 7 NS proteins , NS3 and NS5 are considered as drug targets because they exhibit enzymic activity. NS3 becomes active only if the NS2B protein is also bound to it. In drug design programs NS3/NS2B protein is one of the first targets. NS5 is another potential target . Validated target : It is any protein or cellular or viral component whose inhibition leads to the inhibition of the growth of the virus itself.

13. HETEROCYCLIC COMPOUNDS 2/3 rd of the organic compounds are aromatic heterocycles Majority of drugs used in pharmaceutical and biological industries are heterocycles . Natural drugs : Quinine- was used to prevent and treat maleria in the 16 th century. Synthetic drugs : Antipyrine - first synthetic drug for fever(1887) Heterocycles play an important role in biochemical processes because the side groups of the most typical and essential constituents of living cells are heterocycles . Quinine

14. Recently uncovered DENV INHIBITORS N- sulfonylanthranilic acid derivate Adenosine analogue of (1) chlorophenyl-thiophene derivate (4) 5-methoxyquinazoline- 2,4-diamine

15. A Uracil Based multifunctional compound(fig1)was found to be a potent inhibitor of dengue virus But its De- aza derivative (fig 2)is inactive against the virus Mechanism of action-inhibition of the enzyme, inosine monophosphate dehydrogenase (IMPDH). Other Inhibitors

16. 2,4-Diaminoquinazoline Derivatives Fig(1) Fig(2) Among the derivatives of 2,4-Diaminoquinazoline the one shown in fig (2) has the most efficient antiviral activity It is an inhibitor of the replication of Dengue 2 virus It has a half maximal effective concentration (EC-50) value of 0.05µM It has a low cytotoxicity (50% cytotoxicity concentration)- CC-50 value greater than 100 μ M

17. EXPERIMENTATION METHODOLOGY we focussed on the method of scaffold hopping in drug discovery 2 methyl 4- hydroxy quinoline was the compound selected for modification. synthesis of 1-Phenyl-1,2,3,4-tetrahydro- β -carboline-3-carboxylic Acid following general procedures. Synthesis of hydroxy quinoline using Conrad limpach reaction Substitution reactions of the two compounds

18. CHEMICALS USED L-Tryptophan Benzaldehyde Sulphuric Acid Hydrochloric Acid Ethyl Acetoacetate Aniline Ethanol Petroleum Ether P-toluene Sulphonic acid Silicone oil Dimethyl Formamide (DMF)

19. Reaction of L-Tryptophan with Aromatic Aldehydes to Give Tetrahydro - β - carboline ( 1a-cis and 1b-trans). : PROCEDURE L-Tryptophan(MW=204) was taken (2.94 m.mol) in a round bottom flask It was dissolved in 10 ml of 0.2 N Sulphuric acid To this solution 0.5 ml of benzaldehyde (4.4) m.mol was added and continuously stirred for 9 days. A precipitate was obtained –(245 mg, and 56%) as a white solid. It is a mixture of the cis and trans isomers.

20. The reaction….

21. Synthesis of 2-methyl-4-hydroxy quinoline : Conrad Limpach reaction. In a 100 ml beaker, 0.01 mol, 4.35 mg Aniline, 0.01 mol, 6.35 mg ethyl acetoacetate and 120 mg of p-toluene sulphonic acid were taken and mixed well. This was then stirred in a round bottomed flask which was kept in an oil bath at 100°C. The progress of the reaction was monitored using TLC every 30 seconds. After heating, 10ml Petroleum Ether (60°C-80°C) was added to the mixture. The product was collected by suction and purified by re- crystallisation from DMF-water.

22. Mechanism of Conrad Limpach reaction:

23. Snap Shots

24. Conclusions T he structure of the DENV virus and its mechanism of action was studied and reported Various Heterocyclic compounds were reviewed and their activity against Dengue Virus were listed out. Thus we have made an attempt to synthesize 2 simple heterocyclic compounds Tetrahydro - β - carboline – 3- carboxylic acid 2 -methyl 4-hydroxyquinoline

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