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)

DRUG DISCOVERY METHODSHigh 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!

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

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

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.

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.

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.

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

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

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.

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.

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.

HETEROCYCLIC COMPOUNDS

2/3rd 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 16th 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

RECENTLY UNCOVERED DENV INHIBITORS

(1)N-sulfonylanthranilic acid derivate

(2) Adenosine analogue of (1)(3)chlorophenyl-thiophene

derivate(4) 5-methoxyquinazoline- 2,4-diamine

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

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 activityIt is an inhibitor of the replication of Dengue 2 virusIt has a half maximal effective concentration (EC-50) value of 0.05µMIt has a low cytotoxicity (50% cytotoxicity concentration)- CC-50 value greater than 100μM

EXPERIMENTATIONMETHODOLOGY 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 reactionSubstitution reactions of the two compounds

CHEMICALS USED

L-TryptophanBenzaldehydeSulphuric AcidHydrochloric AcidEthyl AcetoacetateAnilineEthanolPetroleum EtherP-toluene Sulphonic acidSilicone oilDimethyl Formamide (DMF)

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 flaskIt was dissolved in 10 ml of 0.2 N Sulphuric acidTo 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.

The reaction….

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.

Mechanism of Conrad Limpach reaction:

Snap Shots

Conclusions

The 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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