Exploring the Coronavirus Exoribonuclease as an Antiviral Target

Over the last 20 years, three major zoonotic coronavirus (CoV) infections have emerged all causing acute respiratory illness, leading to significant morbidity and mortality. SARS-CoV-1 emerged in Asia in late 2002 while Middle East Respiratory Syndrome (MERS-CoV) was first reported in Saudi Arabia in 2012. In late 2019, SARS-CoV-2 was reported in China and has now spread globally causing over 673,000 deaths in less than eight months. The currently-raging COVID-19 pandemic presents an urgent need to explore new targets and approaches. Many RNA viruses such as Hepatitis C virus (HCV) and Respiratory Syncytial virus (RSV) can be treated with ribavirin and other broad-spectrum antiviral nucleoside analogues. Ribavirin and other nucleoside analogues are misincorporated into progeny genomes by the virally encoded RNA-dependent RNA-polymerase (RdRp), resulting in lethal mutagenesis. Interestingly, ribavirin has minimal effect against SARS or MERS due to unique aspects of CoV replication. The 30kb CoV genomes are the largest of all RNA viral genomes, more than three times the typical size. All CoVs encode 16 non-structural proteins (nsps) required for the production of progeny RNA. Nsp14 contains an exoribonuclease domain (ExoN), which has been shown to ensure replication fidelity of the large genome and provide resistance to drugs like ribavirin. ExoN, a conserved 3' to 5' proofreading exoribonuclease, removes misincorporated ribavirin, rendering it ineffective. Active site mutants of human CoV ExoN result in severe defects in viral RNA synthesis. Thus, ExoN inhibitors are expected to be effective anti- coronavirus agents either as monotherapy or in synergistic combination with nucleoside analogues. This proposal aims to discover first-in-class ExoN inhibitors for further drug development. Our current program is focused on developing broad-spectrum small molecule inhibitors of essential viral exonucleases as antiviral drugs. These exonucleases, like CoV ExoN, possess an acidic active site containing dual magnesium ions that coordinate substrate binding and catalyze bond cleavage. This ExoN key structural motif presents an excellent opportunity to expand our antiviral program to CoVs. We have synthesized hundreds of herpesvirus exonuclease inhibitors tailored to the bi-metallic binding site, which will be evaluated for activity against bacterially expressed ExoN. We propose that inhibitors of ExoN proof-reading activity would exert strong antiviral effects as single agents and would powerfully synergize with ribonucleoside analogs such as ribavirin and remdesivir. Key outcomes of the work would be (1) establishing the druggability of the active site of ExoN for small molecule inhibitors, (2) determining the antiviral activity produced by direct ExoN inhibition, (3) evaluating the potential synergism between ExoN inhibitors and nucleoside analogs and (4) identifying lead candidates for follow on in vivo drug development efforts.