Poster Session - Abstract # 23

Identification of a Coronavirus Macrodomain Inhibitor That Impairs Virus Replication in Cell Culture

Jessica J. Pfannenstiel, Yousef M.O. Alhammad, Joseph J. O’Connor, Anthony R. Fehr

Department of Molecular Biosciences, The University of Kansas, Lawrence, Kansas, USA

Coronaviruses (CoVs) are well-known to emerge from zoonotic sources and cause severe human and veterinary diseases, including recent outbreaks of MERS-CoV and SARS-CoV-2.  All Coronaviruses (CoVs) encode for macrodomain protein (termed Mac1) that binds to and removes ADP-ribose from protein.  Mac1 is essential for pathogenesis, indicating that it is a potential target for anti-viral therapeutics.  Murine-Hepatitis virus (MHV), a model CoV that can be easily studied at BSL-2 levels, is ideal for testing Mac1 inhibitors for their ability to target virus replication because Mac1 is essential for replication of this virus.  However, we lacked high-throughput methods to identify inhibitors of MHV infection.  To create a more efficient drug screening system for MHV, we created a luciferase-expressing virus, which can rapidly measure virus replication.  Previous screens identified a novel compound, MCD-628, that inhibited Mac1 activity in vitro at low micromolar levels.  Interestingly, MCD-628 had no impact on virus replication, and we hypothesize this was due to an acidic side chain that could prevent entry into the cell.  We then tested a compound with an ester at that site, termed MCD-617, and then found that this compound impaired MHV replication by ~ 2-logs without significant cytotoxicity at the concentrations tested.  To validate that MCD-617 specifically targets Mac1, we are passaging virus in the presence of MCD-617 to identify drug-resistant mutants.  Deep sequencing will be performed on drug-resistant mutants to identify any Mac1 or off-target mutations and explore potential mechanistic interactions.