ABSTRACT
Background: The SARS-CoV-2 pandemic has sickened over 245 million people, and has killed more than 5 million worldwide. Recent data proves that vaccinations are highly effective in preventing Covid-19 disease, however antigenic drift and other functional mutations in the virus genome reduce the efficacy of vaccines, indicating that the development of antiviral treatments remain a crucial priority. We report potent antiviral activity against SARS-CoV-2 for a promising, novel class of nitrogen-based heterocyclic compounds. Methods: 232 compounds based on the same class of nitrogen-based hetereocyclic molecules were synthesized to final purity of greater than 99%. This library was screened for antiviral phenotypes in a cytopathic effect (CPE) assay using VeroE6 cells and the SARS-CoV-2 WA1 isolate. Based on the results of the WA1 CPE screen, 47 lead candidates were structurally analyzed, and this information was utilized to design 56 additional compounds. A second antiviral CPE-based screen was performed using these 103 candidates in VeroE6 cells with the SARS-CoV-2 delta variant. Antiviral assays studying SARS-CoV-1 (Urbani) and MERS-CoV were performed in Vero 76 cells utilizing a Neutral Red cytopathic effect assay. Results: Within the same class of structurally related small molecules, we tested an initial set of 232 compounds using a CPE-based assay with VeroE6 cells and the USA/WA1 SARS-CoV-2 isolate. Of the compounds tested, 124 demonstrated potency 10 to 540-times higher than a Remdesivir control tested in parallel. Importantly, we observed no detectable toxicity for the vast majority of these compounds when tested up to a concentration of 30 μ M. The lead candidate in this screen displayed an IC50 of 0.02 μ M and a selectivity index of >1,500. Based on structural analysis of an initial 47 lead candidates, we synthesized 56 new molecules, and tested all 103 in a CPE-based assay using the delta variant, also observing efficacy against this variant of concern. Examples of this same class of compounds also display antiviral activity against SARS-CoV-1 (Urbani) and MERS-CoV in cell-based assays. Conclusion: We have identified a novel class of antiviral compounds with potent activity against SARS-CoV-2. High potency against both the early WA1 isolate and the more recent delta variant, as well as efficacy against SARS-CoV-1 and MERS-CoV, suggest that this class of antiviral compounds has pan-Coronavirus antiviral activity.
ABSTRACT
Background: The novel coronavirus SARS-CoV-2, the causative agent of COVID-19, has caused worldwide social and economic disruption. Initial efforts to treat SARS-CoV-2 were hampered by limited knowledge of the molecular details of SARS-CoV-2 infection. To identify molecular targets for SARS-CoV-2 therapeutics, we mapped the host-pathogen protein interactions of SARS-CoV- 2, and investigated host dependency pathways that are required for SARS-CoV-2 infection using drug, knockdown and knockout screens. Concerns regarding the mutagenic potential of SARS-CoV-2 also led us to inquire whether a conserved set of human host factors may be required for infection by all highly pathogenic coronaviruses, thus representing pan-coronavirus drug targets. Therefore, we also mapped the host protein interactions of SARS-CoV-1 and MERS-CoV. Methods: We cloned, tagged and expressed proteins encoded by SARS-CoV-2, SARS-CoV-1, and MERS-CoV in HEK-293T cells, which are permissive to infection by all three viruses. Cells expressing individual proteins were harvested, affinity purifications performed in 96-well format, and protein mass spectrometry was utilized to identify physical interaction partners of each viral protein. Drug treatments, RNAi knockdowns and CRISPR/Cas9 knockouts were tested for SARS-CoV-2 viral phenotypes in Vero, Caco2 or A549-ACE2 cells. Results: We report 389 high-confidence interactors of SARS-CoV-2, 366 interactions for SARS-CoV-1, and 296 interactions for MERS-CoV. Among the SARS-CoV-2 interactors, we identified at least 66 druggable human proteins or host factors, and screening small molecules targeting these pathways using multiple viral assays have identified at least four sets of pharmacological agents that demonstrate antiviral activity against SARS-CoV-2. Comparison of the host-pathogen interactomes of SARS-CoV-2 with the other highly pathogenic coronaviruses SARS-CoV-1 and MERS highlights shared host interactions which may represent pan-coronavirus drug targets. Conclusion: We successfully utilized systematic protein interaction mapping to identify drug targets for SARS-CoV-2, leading to several Covid-19 clinical studies investigating the efficacy of drugs perturbing these pathways. Furthermore, comparative proteomics of the related coronaviruses SARS-CoV-1 and MERS-CoV identified shared host interactions which may represent pan-coronavirus drug targets. For a full list of contributing authors see: Gordon, D. E. et al. Nature 583, 459-468 (2020);Gordon, D. E. et al. Science 370 (2020).