Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease

Abstract

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease

“…We next used this assay to determine the effect of substrate concentration on initial reaction velocities. We found that, in contrast with all of the other enzymes we have examined in this series of papers [31][32][33][34][35][36], reaction rates did not plateau at higher substrate concentrations but instead continued to increase non-linearly, indicating that nsp15 endoribonuclease behaves as an allosteric enzyme towards the 6 nt U substrate, with a K half of 2140 nM (Figure 3A and Supplementary Figure S2A).…”

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp12/7/8 RNA-dependent RNA polymerase

“…64 compounds were selected as primary hits. Compounds were then discounted if: (i) the compound also scored as a strong hit in parallel HTSs of other SARS-CoV-2 enzymes (with the exception of sulphated naphthylamine derivatives, see below) [47][48][49][50][51][52]; (ii) the compound was reported or strongly predicted to be a promiscuous inhibitor due to colloidal aggregation (LogP value >3.6 and >85% similarity to a known aggregator) [53,54]; (iii) the compound was reported or predicted to be a nucleic acid intercalator. This analysis resulted in the selection of 18 compounds for further validation experiments (Supplementary Table S2).…”

“…Out of these, 46 primary hits were eliminated as they likely represent nonspecific modes of enzymatic inhibition such as colloidal aggregation or interference with the substrate structure. As part of this analysis promiscuous compounds that were identified as hits in other SARS-CoV-2 HTS [46,[48][49][50][51][52] were removed with the exception of five suramin and suramin-like compounds, which were also identified in the SARS-CoV-2 nsp13 helicase HTS (Zeng et al [46]). In vitro validation of the effect of suramin and suramin-like compounds on the activity of SARS-CoV-2 helicase and SARS-CoV-2 RdRp can be found in Zeng et al [46].…”

An all-out assault on SARS-CoV-2 replication

“…Compounds that inhibited viral replication without deleterious effects on uninfected cells were selected as candidate antivirals. The seven papers describing these results are published in this addition of the Biochemical Journal [1][2][3][4][5][6][7].…”

“…The main protease Nsp5 was inhibited by Calpain inhibitor 1 which also reduced viral infectivity in cell culture. Peptidyl fluoromethylketones that mimicked the peptide cleavage site in the virus polyprotein were highly specific and potent inhibitors in vitro but less so in vivo [6]. They represent excellent starting points for further drug development.…”