Discovery of 2-amide-3-methylester thiophenes inhibiting SARS-CoV-2 ADP-ribosyl hydrolysing macrodomain and coronavirus replication

Wazir, S.; Parviainen, T. A. O.; Maksimainen, M. M.; Duong, M. T. H.; Pfannenstiel, J. J.; Cluff, D.; Sowa, S. T.; Galera-Prat, A.; Ferraris, D. V.; Fehr, A.; Heiskanen, J. P.; Lehtiö, L.

doi:10.1101/2023.08.28.555062

bioRxiv · 10.1101/2023.08.28.555062

Discovery of 2-amide-3-methylester thiophenes inhibiting SARS-CoV-2 ADP-ribosyl hydrolysing macrodomain and coronavirus replication

Wazir, S.·Parviainen, T. A. O.·Maksimainen, M. M.·Duong, M. T. H.·Pfannenstiel, J. J.·Cluff, D.·Sowa, S. T.·Galera-Prat, A.·Ferraris, D. V.·Fehr, A.·Heiskanen, J. P.·Lehtiö, L.

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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 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 {micro}M. 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 {micro}M 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.

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Wazir, S., Parviainen, T. A. O., Maksimainen, M. M., Duong, M. T. H., Pfannenstiel, J. J., Cluff, D., Sowa, S. T., Galera-Prat, A., Ferraris, D. V., Fehr, A., Heiskanen, J. P., Lehtiö, L.. 2023-08-29. Discovery of 2-amide-3-methylester thiophenes inhibiting SARS-CoV-2 ADP-ribosyl hydrolysing macrodomain and coronavirus replication. https://doi.org/10.1101/2023.08.28.555062

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