Mechanistic Studies of Small Molecule Entry Inhibitors of Influenza A Viruses

Influenza A viruses (IAVs) are the most problematic among the four genera of influenza viruses owing to their high abilities to genetically adapt to various animal species. Infections caused by IAVs, termed “flu”, lead to seasonal epidemics and have thus far caused 4 large-scale pandemics in the past 100 years. Annual vaccinations against epidemic flu pose problems of reformulation requirements and low efficiencies. A complementary protective measure against influenza infections is the use of antivirals and, among those that are FDA approved, the prevalence of antiviral resistance is greatly concerning. Hence, it is crucial to develop anti-influenza inhibitors that exhibit strong effectiveness against novel viral targets. Considering the criticality of the IAV surface glycoprotein hemagglutinin (HA) for IAV receptor binding and fusion, the focus of this thesis was the following: 1) identification of highly effective and metabolically stable IAV antivirals targeting HA, 2) biophysical characterization of these IAV small molecule inhibitors, and 3) gaining structural insights into the small molecule inhibition of these antivirals. We report the development of highly potent IAV inhibitors, as evaluated by pseudo- and infectious influenza virus assays. Our results also provide strong evidence for the binding of these small molecule inhibitors to HA, proving their mechanism of action to be the stabilization of a neutral conformation of HA. This stabilization will prevent the low pH-dependent irreversible conformational change of the glycoprotein in the late endosomes which is crucial for virus and host organelle fusion. Additionally, we provide data for the assessment of the biophysical properties of protein-drug binding that was done using recombinant HA protein which indicated favorable binding properties based on their kinetic parameters. We also studied the effect of pH on some of these inhibitors and discovered that a decrement in binding affinity (KD) values occur when the pH was reduced from 7.2 to 6.2. Furthermore, using structural analyses, corroborated by in vitro mutagenesis studies, we provide molecular insights into the binding of these small molecule inhibitors to HA, information that will aid the structure-activity relationship studies for further anti-IAV HA-targeting drug development. Our studies focused on one of the two phylogenetic groups of HAs; our group and others have observed that most, if not all, of the HA-targeting small molecule inhibitors possess group-specific activities, and in this thesis, we offer insights into the molecular mechanisms underlying their group-specific inhibition.