ABSTRACT Among the most devastating causes of non-traumatic hemorrhage are the Filoviruses, including Ebola and Marburg. Fatality rates from hemorrhage by infection with either of these viruses can exceed 90%. Their outbreak potential was underscored by the 2013-2016 Ebola epidemic in West Africa, which grew from one infected toddler to over 28,000 cases and claimed ~11,000 lives. In acute filovirus infections, patients often develop endothelial leakage and disseminated intravascular coagulation. This vascular disruption, combined with severe diarrhea and vomiting, can progress to profound hypovolemia, hepatic and renal failure. Ebola and Marburg viruses express a single surface glycoprotein (GP) required for host cell infection. Following macropinocytotic uptake, filovirus infection occurs by GP binding to domain C of the endosomal Niemann-Pick C1 (NPC1-C) receptor. The ZMapp antibody cocktail is one proposed treatment for Ebola virus, but to date there are no approved safe and effective antibody or small molecule inhibitors for any other filovirus infection. To address the need for improved treatment options for these pathogens, I will use structure/function analysis to characterize the mechanism of two promising classes of therapeutic candidates that block receptor binding and are broadly reactive. The first class consists of neutralizing antibodies from a human Marburg survivor that block infection by binding conserved receptor-binding sites on GP. MR191 and MR72 are both potent monoclonal antibodies that target Marburg, but MR72 is unique in its broad filovirus cross-reactivity. Last year I determined the crystal structure of MR191 in complex with Marburg virus GP and grew crystals of a MR72- MARV GP complex. Here, I propose to solve the MR72-GP complex structure, and compare antibody-GP contacts to determine critical interaction sites and how MR72 achieves cross-reactivity. Since MR191 showed lifesaving potential in nonhuman primates and is a possible therapeutic for humans, I will also evaluate MR191 escape mutants, which could be used to assess its potential against emergent viral strains. The second class includes small molecule compounds targeting the NPC1 receptor. I will determine structures of two candidate small molecule inhibitors in complex with NPC1-C, and use Ebola GP and other filovirus GPs pseudotyped onto Vesicular Stomatitis Virus to assess whether these compounds inhibit viral entry. In this focused research plan, I will thus use crystals and reagents already in hand to approach treatment of Ebola and Marburg virus disease from two angles: immunotherapy against the receptor-binding site and small molecule therapy against the receptor. This work will be critical for understanding the inhibitory mechanisms of these ther...