Small molecule inhibitors targeting the SARS-CoV-2 pathogenicity factor Nsp1

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SUMMARY The COVID-19 pandemic has caused great harm to human life and the worldwide economy. Almost 2 years after the emergence of its etiological agent SARS-CoV-2, there are still very few available antiviral drugs. The situation is further aggravated by the emergence of new viral variants that might render available antiviral therapeutics and vaccines less effective in the future. This calls for the development of diverse antiviral strategies, aimed at targeting as many different viral pathways as possible. Here we explore for the first time the possibility to create inhibitors of the viral protein Nsp1 to fight SARS-CoV-2 infection. Nsp1 is a major virulence factor that functions by restricting host gene expression to inhibit antiviral signaling. We have recently identified nine putative inhibitors of Nsp1 function by quantitative high throughput screening. We propose an in-depth structural and functional characterization of the identified compounds to explore their capability to be developed into potent antiviral drugs. In aim 1 we will use NMR spectroscopy to identify the pharmacophore and binding site(s) of the small molecule inhibitors on Nsp1. It is the goal of aim 1 to determine the minimal ligand structural features necessary for Nsp1 inhibition. We will also test commercially available analogs to interrogate which chemical moieties can increase binding and inhibition. In aim 2 we will explore which functions of Nsp1 are targeted by small molecule inhibition. By combining an array of complementary biochemical and cell-based assays, we will interrogate the effect of small molecule inhibition on ribosome binding, mRNA degradation, and mRNA translation. It is the goal of aim 2 to identify the mechanism of inhibition to aid future compound optimization. By pharmacologically targeting selective functions of Nsp1, we might also gain new biological insight into the coronaviral host-shutoff pathway. Overall, these studies should provide insight into the structure and mechanism of potential small molecule inhibitors of SARS-CoV-2 Nsp1, laying the foundation for future chemical optimization of lead compounds with the goal to develop new potent anti-coronaviral drugs.