Abstract

Since the emergence of SARS-CoV-2 in 2019, Covid-19 has developed into a serious threat to our health, social and economic systems. Although vaccines have been developed in a tourde- force and are now increasingly available, repurposing of exis ing drugs has been less successful. There is a clear need to develop new and specific drugs against SARS-CoV-2 that can also be used against future coronavirus infections. We are working on non-structural proteins 1 (nsp1) and 10 (nsp10), two potential antiviral drug targets. Nsp1, a unique viral and conserved leader protein, is a crucial virulence factor for causing host mRNA degradation, suppressing interferon (IFN) expression and host antiviral signaling pathways. Nsp10 is a conserved stimulator of two enzymes crucial for viral replication, nsp14 and nsp16, exhibiting exoribonuclease and methyltransferase activities. Interfering with both non-structural proteins represents interesting intervention strategies. We applied fragment-based screening via X-ray crystallography to identify ligands targeting SARS-CoV- 2 nsp1 and nsp10 located in various novel binding sites. Microscale thermophoresis (MST) and thermal shift (TSA) experiments were used to characterise and quantify fragment affinities for both non-structural proteins. “SAR-by-catalogue” combined with structure-based design led to fragment analogues with improved affinities. Fragments were also investigated for their potential to inhibit nsp1 and nsp10 homologues in two β-coronaviruses of medical relevance, SARS and MERS. These fragments will serve as starting points for the development of more potent analogues using fragment growing, linking and merging techniques combined with structure-based drug design, with the hope that these may also be useful as starting points for future coronavirus outbreaks.