Project One: Small-Molecule Inhibitors of Flavivirus Entry

Dengue virus and other flaviviruses including West Nile virus, tick-borne encephalitis virus, and Yellow Fever virus, are widespread, mosquito- or tick-borne human pathogens. About 40% of the world's population lives in areas with substantial risk of dengue transmission, and as many as 100 million people may be infected annually, experiencing dengue fever and potentially lethal severe dengue. Development of a vaccine effective against all four dengue serotypes has been difficult because infection with one of the four serotypes does not lead to protective immunity against any of the other three and may lead to enhanced risk of severe, life-threatening illness, especially in children. Antivirals against dengue are of interest due to their potential to reduce the severity of dengue-associated disease as well as to reduce transmission. This proposal will contribute to efforts to develop dengue antivirals by developing mechanistically well-characterized small- molecule inhibitors of dengue entry and using these to validate the dengue E protein as an antiviral target in vitro and in vivo. We have previously identified three structurally distinct lead compound series and inhibitors that exhibit a spectrum of activities against dengue virus entry. In the proposed work, we will use a combination of x-ray crystallography, medicinal chemistry, direct protein-small molecule affinity measurements, single-virion fusion and live-cell imaging assays, and resistance studies to optimize the pharmacological activity of our compounds and to elucidate the biochemical and structural mechanisms of action that correspond to potent, pan-serotype inhibition of dengue. Key to these efforts are quantitative assays, analogous to enzymatic activity measurements, for studying the biochemical functions of the viral envelope protein and that allow us to link the biochemical mechanism of small-molecule inhibitors with the step(s) in the cell-entry pathway at which they block infection. Optimized dengue entry inhibitors will be tested in a murine model of dengue infection and pathogenesis. Collectively, our efforts are aimed at producing compounds that can be advanced as preclinical candidates for dengue drug discovery efforts. More broadly, this work establishes the template for a rational approach to the development of small molecule inhibitors of other enveloped viruses as potential antivirals.

The goal of this component of the CETR is to validate the dengue virus E protein as an antiviral target in vitro and in vivo. We have three series of compounds that inhibit dengue virus fusion and hence dengue infectivity. A number of these compounds bind the E protein, probably in the well-characterized pocket at the hinge between domains I and II. We will identify the molecular interactions underlying the effect(s) of these inhibitors on dengue virus fusion, elucidate the biochemical mechanisms responsible for their inhibitory activity, and identify mechanisms of viral resistance. We will use x-ray crystallography, single-particle kinetic studies, live-cell imaging, direct affinity measurements, and studies of resistance mutations in our efforts to optimize the pharmacological activity of our compounds in vitro and ultimately the efficacy of our compoundsin vivo. The experiments are designed to identify pharmacophores with potent, pan-serotype activity and favorable resistance profiles and to correlate these properties with a specific mechanism by elucidating the kinetic intermediate targeted by the inhibitor and by identifying the molecular interactions of the inhibitor with E. This work will lead to one or more compounds suitable for advancement as preclinical candidates.