Discovery of 2-Amide-3-methylester Thiophenes that Target SARS-CoV-2 Mac1 and Repress Coronavirus Replication, Validating Mac1 as an Antiviral Target

Abstract

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed. Here, we describe small-molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation-mediated innate immune responses. Three high-throughput screening hits had the same 2-amide-3-methylester thiophene scaffold. We studied the compound binding mode using X-ray crystallography, allowing us to design analogues. Compound <b>27</b> (MDOLL-0229) had an IC₅₀ of 2.1 μM and was selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human proteins. The improved potency allowed testing of its effect on virus replication, and indeed, <b>27</b> inhibited replication of both murine hepatitis virus (MHV) prototypes CoV and SARS-CoV-2. Sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of <b>27</b>. Compound <b>27</b> is the first Mac1-targeted small molecule demonstrated to inhibit coronavirus replication in a cell model.