Discovery & Characterization of Filo-. Arena -and Alpha Virus Entry Inhibitors

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RNA viruses are prominent members of NIAID's Category A, B, and C lists of biodefense and emerging infectious disease agents. In this proposal therapeutics are being identified, characterized and developed. As our target we have chosen to focus on receptor interaction and entry. This is the first step in establishing an infection, and disruption has proved effective at preventing infection and virus spread. We have developed and published a series of assays that have become a platform technology for identifying such drugs and, importantly, for mechanism of action analysis. Our systems are now developed to the point where we can perform very high-density screens. To begin, we will use a library of 200,000 compounds in collaboration with the NIH Chemical Genomics Center. Active compounds will be prioritized based on comparative analyses of structure-activity relationships across the panel of assays determined from multiple dosages. Our UTMB team will work to develop hits into useful drugs. We will systematically compare two members each of three classes of virus, the filoviruses, Ebola and Marburg; arenaviruses, Lassa and Junin; and the alphaviruses, Venezuelan equine encephalitis and chikungunya viruses. Comparing each virus in one screen will be an effective means to identify potential drugs active against 1) specific virus types, 2) virus families, or 3) those of broad spectrum activity. All drugs will be tested against wild-type viruses. Next, we will follow up on our Ebola virus study, in which we identified FDA-approved drugs effective at blocking Ebola virus infection. In preliminary screens with small drug libraries and siRNA, we discovered that drugs that block calcium flux into cells are effective at inhibiting Ebola virus infection and cell entry, thereby preventing cytopathic effect. We will characterize each drug's mechanism of action. First, we will identify the most potent calcium channel blocking drugs via our specialized virus entry assays and use of wild-type virus. Second, we will address mechanism of action by identifying downstream signaling targets (e.g., CALM and CAM kinases) that may, in turn, be useful targets for anti-viral therapy. Third, we will examine the role of virus envelope protein in triggering this cascade by biochemically analyzing envelope protein-cell interactions. Finally, we will test the candidate drugs in animal disease models. We will also test other filoviruses, including Marburg virus. Our findings will aid in developing a robust filovirus therapy and an understanding of how calcium signaling functions in virus infection and potentially pathogenesis.