Discovery of 2-amide-3-methylester thiophenes that target SARS-CoV-2 Mac1 and repress coronavirus replication, validating Mac1 as an anti-viral target

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 to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 μM. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 μM and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of both MHVa prototype CoV, and SARS-CoV-2. Furthermore, sequencing of a drug-resistant MHV identified mutations in Mac1, further demonstrating the specificity of 27. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts.

. Assay validation statistics.Table S2.Hit validation and counter screening.Table S3.Crystallography data and refinement statistics.

Figure S1 .
Figure S1.Representative IC50 measurements of SARS-CoV-2 Mac1 for ADPr and initial hit compounds.Measurements were carried out in half-logarithmic dilution starting from 100 to 0.003 µM for each curve.Negative control (containing no compound) was set one logarithmic unit below the lowest concentration.positive control (containing 200 µM ADPr) was set one logarithmic unit above the highest concentration of the compound.Data is reported as mean ± SD of four replicates.

Figure S3 .
Figure S3.Compound 27 does not affect the viability of mouse or human cell lines.(A-B) L929 (A), DBT (B) and Calu-3 (C) cells were treated with compound 27 at the indicated concentrations for 24 hours and then viability was measured by an MTT assay and normalized to the viability level of DMSO treated cells.The results in A and B are representative of 3 independent experiments, while the results in C are the combined results of 3 independent experiments.n=3.
*Calculated from Z' values.

Table S2 .
Hit validation and counter screening results with TNKS ARC4 assay.

Table S3 .
Data collection and refinement statistics for co-crystal structures.