Studies of Small Molecule Inhibitors Targeting the Filovirus Glycoproteins and Potential Therapeutics

Filoviridae, including Ebola (EBOV) and Marburg (MARV) viruses, are emerging pathogens that pose a serious threat to public health. Continuous outbreaks in Africa and the discovery of novel filovirus pathogens in other continents like Měngla (MLAV) in China highlight the urgency for the development of therapeutics against novel and existing filovirus pathogens. To date, no therapeutics have been approved to treat filovirus infectious, representing a major unmet medical need. The overall goals of this thesis are to develop broad-spectrum antivirals targeting the viral glycoprotein (GP) and to elucidate the mechanisms of small-molecule inhibition. Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. This thesis focuses on 1) the discovery of pan-filovirus entry inhibitors compounds 30 and 32, 2) the mechanism of pan-filovirus entry inhibition and 3) the elucidation of potential therapeutics and entry mechanism for MLAV. First, we performed a focused screen on estrogen receptor ligands leading to the discovery of tamoxifen analog ridaifen-B as a potent entry inhibitor of EBOV and MARV. Optimization and reverse-engineering to remove ridaifen-B’s estrogen receptor activity led to the creation of novel pan-filovirus inhibitors 30 and 32. Second, we identified the HR2 region as the primary binding site of MARV and the secondary binding site of EBOV entry inhibitors confirming that small-molecule entry inhibition is multi-mechanistic. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinct mechanisms of action and lay the groundwork for future development of anti-filoviral agents. Finally, we provide evidence that the previously identified small molecules and neutralizing antibodies known to inhibit EBOV and MARV entry also inhibit MLAV.Our findings support that MLAV is more closely related to MARV than EBOV, yet distinctive from MARV, which is consistent with the previously published studies. Overall, the findings presented in this thesis provide important insight into filovirus entry and rational drug design of pan-filovirus drug development.