Sandeep S.

Raut

Roll No. 1120107

Suggesting a Chaperone inhibitor as antiviral drug in order to overcome the problem of resistant escape mutant viruses Introduction: The prevention and cure from viral infections is always a big challenge in front of medical science. Though, many antiviral drugs are available in the market, they are not ever potent to combat against the outbreak of drug resistant escape mutants. Thus, designing an antiviral drug targeting the viral component does not seem to be potent always, as many viruses always reattack with slightly modified genetic make-up and thus with novel antigenic determinants. Hence, to find the consistent target for anti-viral drug is a crucial deal. Recent investigations suggest the important role of chaperones in viral replication cycle. Many chaperones act as host factors involved in viral RNA replication suggesting that these viruses use established cellular chaperone pathway to assemble its RNA replication complex on intracellular membrane. Many chaperones are identified as essential factors in the folding and maturation of viral capsid proteins as well as RNA dependent RNA polymerase. Viral replication and progeny assembly take place exclusively in the cytoplasm of infected cells at discrete foci enriched in virus RNA and proteins termed viral factories or virosomes and many co-localized cellular chaperones are found to be the important players to assemble such virosomes. This suggests the possible application of chaperone inhibitor as ever potent antiviral agents. Pharmacologic inhibition of such chaperones must impair the viral replication in cell culture. Apart from this, unlike other antiviral drugs, chaperone inhibitors will reduce the viral replication without emergence of drug resistant escape mutants. Pharmacogenomics provides an access to find drugs (inhibitors) against these chaperones. This approach includes… (i) Inhibition of host chaperone (ii) Depletion of viral client protein (iii) Simultaneous inhibition of one of the steps in viral replication cycle (iv) Antiviral effect and therapeutic index. Consider an experimental query in order to find such a virosome associated chaperone and hypothesize an antiviral drug against such a positive sense RNA virus in that case. Materials and Method: A] Materials: (1) Target cells (cell culture) (2) Stock of ‘+’ sense RNA virus (3) Various antibodies: Against different chaperones, against the whole virion, against viral proteins (4) Inhibitors for few chaperones B] Methods: (1) Preparation of virus infected cell extracts (2) Western blot analysis (3) Affinity chromatography (4) Conventional immunoflourescence microscopy

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P. P-/. N. which has been assumed to be target for antiviral drug design. For this purpose(1) Make double mutant strains M-/-.cells do not harm the normal cellular process. N. which are as follows(1) Distinguished Chaperones (M-R). (2) Take strains M-/-. (2) Chaperones co-localized with nucleus. Expected result: Let us assume that the chaperones M. O.and R-/. The antiviral response may be because of many possibilities.and R-/. P and R are found co-localized with virosome. Q and R are found to be over expressed. b) Focus on the strains which show reduced or no viral replication. B] Process the cells for confocal immunoflourescence microscopy with anti-chaperones detected with different flourophore conjugated secondary antibodies. N-/-.and R-/.and R-/-.and proceed the experiment with P-/. Observe the culture for normal cell proliferation. and thus shortlist the chaperones to be focused in the experimental query. Expected result: Let us reject strain M-/. O. a) Reject the double mutant for which there is the normal viral response as like that of the given cell culture. Cellular and Viral Genomes will be stained differently. 1120107 (5) Confocal laser scanning microscopy (6) Gene expression microarray. Experiment Design: A] First quantify the microarray based over expression of the chaperones due to viral infection. P-/. Expected result: Let us assume that chaperones M. N. Expected result: Let us assume M-/-. P. (3) Chaperones co-localized with virosome.strains. residing neither in the nucleus nor in virosome/virus factory. Q and R as well as for cellular and viral genomes. But we can check for two major possibilities which are as follows If the targeted chaperone is associated with folding of early viral proteins like RNA dependent RNA polymerases. Raut Roll No. where as Chaperone N is found to be essential for certain cellular process which is vital for cell survival.and infect them with the given positive sense RNA virus and observe for the response. Three possible results may come out of the above mentioned experiment. P-/. C] Verify the involvement of co-localized Chaperones in viral replication and also verify that inhibition of our target chaperone does not harm normal cell proliferation. there will be no or very less viral RNA replication which Page 2 of 4 . Images are taken for chaperones M.Sandeep S.

Let us deal with the 2nd possibility where there is accumulation of viral RNA. Later these suggested inhibitors as a drug needs to undergo preclinical and clinical development.  If the targeted chaperone is associated with Ribosome Associated Complex (RAC). select Chaperone R to be the target for Drug design. we will choose some of the chemical inhibitors which target the functional domain of the chaperone. And. This way. as the major effectors.Sandeep S.00 Thus.00 41. 1120107 can be detected by counting the number of virions and comparing this with the control these chaperones are working fine. there will be no translation of viral proteins. Page 3 of 4 . In this case. Expected result: Strain P-/R-/Fold accumulation of viral RNA than normal 8. D] Drug Designing: Using in-silico approaches we can design various chemical inhibitors (drugs) against the finally selected chaperone on the basis of drug-chaperone interaction using various docking tools and software. there will be many fold accumulation of viral RNA which is not being translated. combinatorial chemistry and genomics. These include high throughput screening. further wet lab trials with those drugs will enable us to finalize the drugs which will inhibit the cellular chaperones without being toxic to normal cells but viruses. Raut Roll No.8-42.8-11.

Francis Burrows2.‡. U. 1120107 REFERENCES (1) Redistribution of Cyclophilin A to Viral Factories during Vaccinia Virus Infection and Its Incorporation into Mature Particles Ana Paula V.2. William P.* (10) Evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance Ron Geller1.1 Técia M.∗ and H´el`ene Sanfac¸on2 (5) Pharmacogenomics in cancer drug discovery and development: inhibitors of the Hsp90 molecular chaperone Paul Workman. Shield2. Kampmueller1 and David J. Len Neckers3 Neal Rosen4 (8) Development and application of Hsp90 inhibitors David B. Che-Sheng Chung and Wen Chang (4) Cellular Remodeling During Plant Virus Infection Jean-Franc¸ois Lalibert´e1.4 Page 4 of 4 . A. Miller1. Raul Andino2. Craig3. Solit1 and Gabriela Chiosis2 (7) Drugging the cancer chaperone HSP90: Combinatorial therapeutic exploitation of oncogene addiction and tumor stress Paul Workman1. Weeks1. and Judith Frydman1.2 Nissin Moussatché. Miller1. Castro. Marco Vignuzzi2.3. Raut Roll No.Sandeep S.* (3) Molecular Chaperone Hsp90 Is Important for Vaccinia Virus Growth in Cells Jan-Jong Hung.2. Damaso1 * (2) A Targeted Analysis of Cellular Chaperones Reveals Contrasting Roles for Heat Shock Protein 70 in Flock House Virus RNA Replication▿ Spencer A. Chandan Sahi3. Elizabeth A.1 and Clarissa R. Carvalho. Solit1 and Gabriela Chiosis2 (9) The Cellular Chaperone Heat Shock Protein 90 Facilitates Flock House Virus RNA Replication in Drosophila Cells Kathryn M. Sabine Rospert4 and David J. PhD (6) Development and application of Hsp90 inhibitors David B.