Targeting Viral Proteins and Host Factors for Discovery and Development of Antivirals Against Influenza, Enterovirus, and Coronavirus
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The University of Arizona.Rights
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Release after 08/10/2023Abstract
There is urgent and continuous need for the next generation of antiviral drugs with a novel mechanism of action due to the limited therapeutic options and increasing drug-resistance. Influenza viruses are respiratory pathogens that are responsible for seasonal influenza and sporadic influenza pandemic. The therapeutic efficacy of current influenza vaccines and small molecule antiviral drugs is limited due to the emergence of multidrug-resistant influenza viruses. Due to the essential function in viral replication and high sequence conservation among influenza viruses, influenza polymerase PA-PB1 protein-protein interaction becomes an attractive drug target. To identify influenza PA-PB1 interaction inhibitors, we have developed a robust high throughput screening method using split luciferase complementation-based (SLC) assay specifically for screening inhibitors of influenza polymerase PA-PB1 interaction and identified two PA-PB1 interaction inhibitors, R160792 and R151785, with potent and broad-spectrum antiviral activity against a panel of influenza A and B viruses, including amantadine-, oseltamivir-, or dual resistant strains. Mechanistic study reveals that R151785 inhibits PA nuclear localization, reduces the levels of viral RNAs and proteins, and inhibits viral replication at the intermediate stage, all of which are in line with its antiviral mechanism of action. As the AM2-S31N mutant persists in more than 95% of current circulating influenza A viruses, targeting the AM2-S31N proton channel appears to be a logical and valid approach to combating drug resistance. We have identified an AM2-S31N inhibitor UAWJ280 with in vivo antiviral efficacy in mice that are infected with either oseltamivir-sensitive or oseltamivir-resistant influenza A strains, and it has a synergistic antiviral effect when combined with oseltamivir. The enterovirus genus of the picornavirus family contains many important human pathogens. Enterovirus D68 (EV-D68) primarily infects children, and the disease manifestations range from respiratory illnesses to neurological complications such as acute flaccid myelitis (AFM). Enterovirus A71 (EV-A71) is a major pathogen for the hand, foot, and mouth disease (HFMD) in children and can also lead to AFM and death in severe cases. Coxsackievirus B3 (CVB3) infection can cause cardiac arrhythmias, acute heart failure, as well as type 1 diabetes. There is currently no FDA-approved antiviral for any of these enteroviruses. Through phenotypic high throughput screening using EV-D68 CPE assay, we identified pyrazolopyridine-containing compound that has moderate antiviral activity. SAR studies yielded several lead compounds which inhibited different strains of EV-D68, EV-A71, and CVB3 with nanomolar range EC50 values and high cellular selectivity indexes. Serial viral passage experiments, coupled with reverse genetics and thermal shift binding assays, suggested that these molecules target the viral protein 2C. Next, we will utilize the best lead compound Jun5-7-1 as a chemical tool to characterize the biochemical functions of Enterovirus 2C protein and validate 2C protein as an antiviral drug target. There are seven human coronaviruses: SARS-CoV, MERS-CoV and SARS-CoV-2 which cause severe acute respiratory syndrome; and four common human coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1 that cause a significant portion of upper and lower respiratory tract infections in humans worldwide. As the third coronavirus outbreak in human history, the COVID-19 pandemic is a timely reminder of the urgent need of broad-spectrum antiviral drugs that can be rapidly deployed for the prevention and treatment of emerging and re-emerging viral diseases. To this end, we have established two cell culture based antiviral assays, cytopathic effect (CPE) assay and plaque assay for HCoV-229E, HCoV-NL63, HCoV-OC43, to expedite the discovery of new antiviral compounds against coronaviruses in biosafety level 2 facilities and stockpile broad-spectrum antivirals against current circulating as well as future emerging coronaviruses. We also characterized the broad-spectrum antiviral activity of lactoferrin (LF) and brilacidin against multiple common human coronaviruses including HCoV-OC43, HCoV-NL63, and HCoV-229E as well as SARS-CoV-2 pseudovirus and their mechanism of action were elucidated. Both LF and brilacidin were found to bind to Heparan sulfate proteoglycan (HSPGs) on the host cell surface, thereby preventing viral attachment to the host cells, as evidenced by 1) drug time-of-addition experiment suggests that both LF and brilacidin exerts antiviral activity at the attachment stage; 2) LF and brilacidin inhibits viral attachment and the inhibition was diminished in the presence of heparin; 3) heparin decreased the potency of LF and brilacidin in cell culture. These studies support the translational potential of LF and brilacidin as broad-spectrum antivirals for coronaviruses including SARS-CoV-2.Type
textElectronic Dissertation
Degree Name
Ph.D.Degree Level
doctoralDegree Program
Graduate CollegePharmacology & Toxicology