Project 6 - Development of Antivirals against Alphaviruses

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Alphaviruses are enveloped plus-sense RNA viruses that include a number of important human pathogens such as the arthritogenic alphaviruses chikungunya virus (CHIKV), Mayaro virus, and Ross River virus, and encephalitic alphaviruses such as Eastern and Venezuelan equine encephalitis viruses. These viruses have emerged as world-wide public health and/or biodefense threats, but to date there are no licensed vaccines or antiviral therapies. Project 6 in the AC/DC seeks to develop orally available direct-acting antivirals against alphavirus infection. We focus on targeting the RNA replication complex, which is formed by the coordinated activities of the four alphavirus non-structural proteins (nsP1-4). Promising preliminary data with our existing chemical assets demonstrate inhibition of CHIKV RNA replication by nucleosides and by inhibitors of the essential protease activity of nsP2. Moreover, prophylactic treatment of mice with our ribonucleoside EIDD-2749 prevented CHIKV viremia and disease. We will build on these findings through the following Aims, in close collaboration with the AC/DC Cores: 1. Optimize and characterize inhibition by the previously identified nucleoside inhibitors EIDD-2749, EIDD- 2997, and additional prodrugs/analogs developed from AC/DC SAR studies. We will define their mechanism of action using our panel of available protein, cell-based, and virus infection assays. We will determine the breadth of inhibition across other alphaviruses and determine resistance profiles and effects on virus replication in cell culture. 2. Perform high throughput screening for inhibitors of CHIKV RNA replication using a cell-based replicon reporter system. Hits will be progressed to preclinical development in collaboration with Cores B and C, and mechanisms defined as in Aim 1. 3. Optimize and characterize inhibitors of the nsP2 protease. We will optimize and further develop our initial inhibitors of nsP2 protease, and use a cell-based screen to identify additional nsP2 protease inhibitors. We will define their mechanisms by using cell-free nsP2 enzymatic assays and virus infection, as well as by applying the strategies described in Aims 1 and 2. 4. Characterize in vivo efficacy. We will use established mouse models to determine the in vivo efficacy of EIDD-2749 and early leads from Aims 1-3 against acute and chronic CHIKV infection and disease. We will also develop a novel reporter mouse line with an integrated alphavirus minigenome template that can detect alphavirus-mediated RNA replication with high specificity and sensitivity. This strategy will be used to follow infection by unmodified alphaviruses, to identify target cells during the acute and chronic phases of CHIKV infection, and to evaluate antiviral therapies developed in this proposal.