Development of direct-acting flavivirus inhibitors

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ABSTRACT Development of potent and broad-spectrum direct acting antivirals (DAAs) is key as a first-line defense against diseases caused by flaviviruses (e.g., dengue virus, yellow fever virus, West Nile virus, Japanese encephalitis virus, Zika virus and tick-borne encephalitis virus). To promote global health, the underlying premise of this proposal is to combine expertise and cutting-edge technology of academic and pharmaceutical sectors to establish a robust environment for drug identification and commercialization. The envisioned development process of broad-spectrum DAAs against flaviviruses is divided in three stages (aim 1–3). The first aim entails the identification and validation of conserved flavivirus targets and hits (driven by Discovery Core B and the Enzymology Core C. Already identified RNA-dependent RNA polymerase (RdRp) hits will serve as the chemical starting point for further optimization by MedChem Core D. In addition, in collaboration with the Discovery Core B, we aim to identify novel structurally conserved binding pockets of the following key viral replication enzymes: nonstructural protein 5 (NS5) RdRp, NS3 helicase, and NS2B-NS3 protease. Compounds targeting those sites will be explored using virtual docking, targeted small-molecule and fragment-based high- throughput screens and DNA-encoded chemical library (DEL) screens. Target sites will be validated in enzymatic assays. We will determine the role of key amino acids in enzyme activity, viral replication and fitness through protein engineering. At least two to four hit series will then be optimized and tested in aim 2 (Hit to Lead). Compounds will be characterized for activity in biochemical and cell-based viral replication assays, for favorable in vitro drug absorption, distribution, metabolism, excretion and toxicity (ADME-TOX), mechanism of action and resistance. Our heterogeneous flavivirus panel will assess the level of broad-spectrum antiviral activity in diverse cell types. While we aim to develop broad-spectrum anti-flavivirus drugs, several flaviviruses cause significant human disease globally, so highly selective inhibitors will also be pursued as appropriate. Lead compounds will be shared with Projects 1, 2, 3, and 5 to evaluate breadth across other viral families. At least two lead compounds selected in aim 2 will be tested in aim 3 for in vivo efficacy. After proof of concept in an in vivo study, we anticipate to deliver one Flavivirus inhibitor active against at least one flavivirus, or at best against all flaviviruses described in this proposal, ready to start Investigational New Drug Application (IND)/ Clinical Trial Authorization (CTA)-enabling studies, and subsequently if approved, enter clinical development and start Phase1 studies.