DESCRIPTION: Ebola viruses (EBOV) and Marburg virus (MARV) are two genera of enveloped viruses that constitute the family of Filoviridae. Outbreaks of fulminant hemorrhagic fever in human and non-human primates in Central Africa caused by EBOV have reached mortality rates of up to 90% in the past and the mortality rate in the recent West Africa outbreak is 60%. No specific antiviral treatment or vaccine is approved for these deadly pathogens. There is thus an urgent need to develop effective antiviral therapies against infection by filoviruses. Targeting cell entry of enveloped viruses as an antiviral strategy has been proven effective against a wide range of viral diseases. However, the intracellular sequestration of filovirus fusion machinery makes it challenging to develop antivirals that block EBOV glycoprotein (GP)-mediated viral entry. We aim to overcome this challenge by adding a cell penetrating peptide sequence and conjugating a lipid moiety to fusion inhibitory peptides (C-peptides). We have shown that sustained plasma levels of our lipid-conjugated EBOV C-peptide are achieved after parenteral administration in mice. This C-peptide inhibits in vitro infection by EBOV with an IC50 of 0.2 ?M and efficiently protects mice from lethal EBOV infection. We propose to leverage these preliminary results to design, synthesize and evaluate novel EBOV C-peptide analogs that can be delivered intranasally or subcutaneously, and have enhanced efficacy against a broad range of filoviruses. Analogs will be evaluated for antiviral activity in infectivity assays, for cytotoxcity on human cells, and for GP2 subdomain-binding interactions. Promising analogs will be evaluated for in vitro antiviral activity against diverse filoviruses and for toxicity in mice. We ill also identify the molecular determinants of antiviral resistance. Selected analogs will be tested in challenge experiments in a mouse model. We anticipate that the knowledge gained from our proposed studies will significantly enhance our ability to address the threat of natural and intentional epidemics by developing potent antiviral drugs with feasible delivery routes for containing acute filovirus outbreaks. We propose the following specific aims: Aim 1. To use structure-guided mutagenesis and protein engineering to optimize the antiviral potency and bioavailability of EBOV C-peptide inhibitors. a) Design and synthesis of new C-peptide analogs; b) Characterization of antiviral activity against a range of filoviruses in cell culture; c) Characterization of determinants of viral resistance by in vitro virus evolution experiments. Aim 2. To evaluate the protection afforded by novel EBOV C-peptide inhibitors delivered intranasally or parenterally against lethal EBOV infection in mice. a) Analysis of in vivo biodistribution of improved EBOV C-peptide analogs; b) Evaluation of toxicity in mice; c) Assessment of in vivo potency and breadth of activity of fusion inhibitors in the mouse model of EBOV infection.