Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease

Abstract

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication. SARS-CoV-2 proliferation relies on the enzymatic activities of the non-structural proteins (nsp), which makes them interesting targets for the development of new antiviral treatments. With the aim to identify novel SARS-CoV-2 antivirals, we have purified the exoribonuclease/methyltransferase (nsp14) and its cofactor (nsp10) and developed biochemical assays compatible with high-throughput approaches to screen for exoribonuclease inhibitors. We have screened a library of over 5000 commercial compounds and identified patulin and aurintricarboxylic acid (ATA) as inhibitors of nsp14 exoribonuclease in vitro. We found that patulin and ATA inhibit replication of SARS-CoV-2 in a VERO E6 cell-culture model. These two new antiviral compounds will be valuable tools for further coronavirus research as well as potentially contributing to new therapeutic opportunities for COVID-19.

Activation of the SARS-CoV-2 NSP14 3′–5′ exoribonuclease by NSP10 and response to antiviral inhibitors

“…At the writing of this publication, numerous publications have highlighted the difficulties encountered expressing full-length (FL) NSP14 in quantities required for structural studies in the absence of fusion peptides or coexpression with other NSP proteins. As noted by other studies, we were also unable to coexpress NSP10 and NSP14 in stoichiometric or near stoichiometric amounts ( Supplemental Figure S1 )( 11 , 16 , 17 ). Additionally, tethering two proteins on a single polypeptide can generate artificial or non-biologically relevant complex interactions or altered domain interfaces.…”

“…Additionally, tethering two proteins on a single polypeptide can generate artificial or non-biologically relevant complex interactions or altered domain interfaces. Structural and enzymatic studies of NSP10/14 and other protein complexes can be significantly altered by large expression tags and nonstoichiometric coexpression of complexes, as demonstrated in ( 17 ). Overall, the lack of straightforward purification protocols of NSP10 and NSP14 have hampered biochemical and structural studies of this important SARS-CoV-2 complex.…”

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase

“…We showed that this compound has an IC 50 of ∼9 mM and is not detergent-sensitive in in vitro assays. FPA-124 was not a hit in any of the other screens in this series [41,47,[51][52][53][54] providing further evidence for its nsp13 specificity. When tested in cell-based assays, FPA-124 inhibited viral growth at an EC 50 of ∼14 mM, below its observed cytotoxicity (>100 mM).…”

“…Nonetheless, our experiments in Vero cells showed no synergistic inhibition on viral replication when combining remdesivir and our nsp13 inhibitors. It would be of interest to test the potential synergy between different nsp13 inhibitors and also between nsp13 inhibitors and SARS-CoV-2 enzyme inhibitors identified in parallel screens [41,47,[51][52][53][54] in the future. Recent studies on host-virus interactions suggest that SARS-CoV-2 nsp13 may also be implicated in immune suppression as it targets several host proteins involved in innate immune signalling pathways such as the interferon pathway and NF-κB pathway [72].…”

“…Notably, a kinase inhibitor, FPA-124, inhibited nsp13 with a micromolar IC 50 of ∼9 mM, and its IC 50 value was not affected by the addition of the non-ionic detergents Tween-20 or Triton X-100 (Supplementary Figures S8 and S9), suggesting its nsp13 inhibition was unlikely to be due to aggregation. Moreover, FPA-124 did not score as a hit in the RdRp screen nor parallel screens against other SARS-CoV-2 enzymes [41,47,[51][52][53][54], indicating its inhibitory activity is likely target specific. Examples of the observed IC 50 curves are shown in Figure 4.…”