New Insights into the Mechanisms of Antiviral Resistance and Action

Abstract

Due to the error-prone nature of viral replication, antiviral drugs can select for drug-resistant strains. Understanding the determinants for drug resistance is useful for the preclinical evaluation of antivirals, and provides important insight into the biochemical nature of the target. The first part of this dissertation describes studies designed to investigate properties of antiviral drug resistance using a well-established drug target from the influenza A virus as a model protein. The second part of the dissertation mainly focuses on the validation of a novel drug target for a re-emerging virus using biochemical and viral replication assays. Influenza A viruses are pernicious human pathogens that lead to seasonal and pandemic outbreaks. Amantadine and rimantadine are two FDA-approved drugs that target a viral proton channel called M2. These two therapeutics are no longer used due to the emergence of drug resistance. The two principal mutations that give rise to resistance in circulating strains are the S31N and V27A found within the pore of M2. Structural characterization of M2 has guided efforts to target these individual drug resistant channels through the conjugation of the adamantane group, and it was shown that the best V27A inhibitor has in vivo activity, and that the best S31N inhibitor displayed favorable pharmacokinetic properties. Although the determinants of drug resistance for amantadine and rimantadine have been thoroughly investigated, similar studies have not been performed for inhibitors of the V27A and S31N variants. Due to the fine-tuned function and structure of M2, I have hypothesized that V27A and S31N inhibitors would have a high genetic barrier to drug resistance. Using representative V27A and S31N inhibitors, I performed serial passage experiments in cell culture to select for drug-resistant influenza A viruses. Sequencing of the M2 gene segment identified mutations in the channel that gave rise to resistance, which were confirmed using recombinant viruses. In collaboration, functional assays and molecular dynamics simulations were performed to characterize the biophysical and biochemical properties of each mutation. Overall, I discovered that there are at least three mutational strategies for resistance that are selected: 1) mutations in pore-lining residues, 2) mutations in residues lining the interhelical region, and 3) mutations in the C-end of the channel below the tryptophan gate. Analysis of each mutation reveals unique effects on viral fitness, channel function, structural flexibility, pore hydration, and pore size. These studies reveal the utility of cell culture passage experiments in evaluating drug resistance, and provide insights into the mutational landscape of M2 in actively replicating influenza A viruses. The second part of this dissertation mainly focuses on the validation of a drug target from enterovirus D68, a re-emerging virus linked with neurological disease in children. No antivirals are currently available for EV-D68 infections, and few viral proteins have been validated as drug targets. I performed sequence alignment analysis which suggested that the D68 2A gene segment may encode a viral cysteine protease, and may therefore represent an attractive target for antiviral development. To test this hypothesis, we developed a protein expression protocol to obtain the protein for functional experiments. Using a fluorescence resonance energy transfer (FRET)-based assay, the expressed 2A protein was used to confirm and characterize the protease activity. I subsequently identified telaprevir—an FDA-approved peptide mimetic used to treat HCV infections—as a potent inhibitor of enterovirus D68 by targeting the 2A protein. By using drug resistance selection, I was able to confirm the antiviral activity by telaprevir was due to 2A inhibition. The enteroviral replication assays I utilized for testing telaprevir activity were further applied to study other enteroviruses and protein targets. Overall, by using a combination of biochemical functional assays, cellular antiviral assays, and molecular modeling, we have provided new insights into the mechanism of action and resistance for novel antiviral targets.