Functional studies of antivirals targeted against the dengue virus envelope protein

Mansoora Khaliq, Purdue University

Abstract

Flaviviruses are small, positive sense RNA viruses, which are responsible for causing many serious human diseases such as yellow fever, dengue, and West Nile. Dengue virus is the causative agent of dengue fever and the virus infects 50-100 million people per year with a mortality of greater than 25,000. Currently there is no approved vaccine nor are there any antivirals against the virus. This study looks at the biological evaluations of compounds designed and synthesized which are targeted against the dengue envelope protein. The crystal structure of the ectodomain of the dengue-2 envelope (E) protein has previously been solved revealing a hydrophobic pocket located at a hinge region between domains I and II of the protein. This pocket was found to have the detergent N-octyl-β-D-glucoside (BOG) bound in at least one crystal form of the protein. With the aid of an in-silico screen, compounds from the NCI small-molecule library were screened for predicted binding in the hydrophobic pocket. Initially, the top identified compounds were evaluated against replication of virus, in which one compound 111056 was identified as a potent inhibitor of virus. Binding of a small molecule such as 111056 in the pocket may affect the virus mainly because this pocket serves the role of a hinge, and changes angles during transitional phases of the virus life cycle such as entry and maturation. From our previous results, NMR spectroscopy has identified this compound as one which competes with BOG for binding in the hydrophobic pocket. Characterization of the compound showed that it inhibits dengue replication at a concentration of 30 μM, reducing the yield of infectious virus by more than 10-fold. Time of addition experiments reveal that this compound has an effect against dengue virus entry, in addition to a later step in replication. This compound also affects dengue virus adsorption onto cells, and does so specifically by binding to the virus, and not the cellular receptors. Further analysis of the compound's effect against viral entry through low pH-mediated fusion has identified that the compound is able to prevent fusion of cells at 20-30 μM concentration. Through mass spectrometry data, we have identified critical residues Ser 274, Gln 271, and Thr280 on the dengue E protein which were initially predicted, but are now shown to be involved in direct interaction with the compound 111056. Other contact points of the compound leading to an adsorption defect are with residues Asn153 and Asn67. Second and third generation compounds have been synthesized and biologically evaluated. The top second generation compound, 36, contains a central thiazole core, which is necessary for its antiviral activity. Characterization of the compound against dengue has revealed that the compound affects entry, but is a more potent replication inhibitor. Third generation compound synthesis has focused on the structure of compound 36, focusing on changes of moieties at the C4 and C5 position representing the dibromo groups and the unstable methyl ester group respectively. Results thus far have shown that a change at C5 to a methyl thioester, and a change at C4 to a monobromo moiety gives stability and also produces an enhanced therapeutic index for the compound.

Degree

Ph.D.

Advisors

Kuhn, Purdue University.

Subject Area

Biology|Virology

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